CN110881570B - Feed additive and feed containing same - Google Patents

Abstract

A feed additive and feed containing the same can enhance animal immunity, promote vaccine immunity, and resist pathogenic bacteria infection and obesity. The feed additive of the invention comprises: a bacterial extract comprising a plurality of bacterial inclusions, wherein the bacterial inclusions comprise a polypeptide comprising: a translocation peptide for translocation; and a binding epitope.

Description

Feed additive and feed containing same

Technical Field

The invention relates to a feed additive and a feed containing the same, in particular to a feed additive capable of enhancing animal immunity and improving vaccine immunity effect and resisting pathogenic bacteria infection and obesity and a feed containing the same.

Background

In the livestock raising process, in order to make the livestock grow more lean meat and less intramuscular fat, good price is sold, and lean meat extract is added by the bad breeder to improve the lean meat percentage of the livestock. However, clenbuterol is a type-B sympathetic receptor agonist, and when people eat meat containing clenbuterol, adverse effects such as acceleration of heartbeat and the like can be caused, and particularly, the influence on patients suffering from hypertension or heart disease is more remarkable; so the addition of clenbuterol as a feed additive is now prohibited. Therefore, if a feed additive capable of replacing clenbuterol without the adverse effects described above could be developed, it would significantly contribute to the raising of livestock.

Chicken newcastle disease (commonly known as fowl plague) and avian influenza (abbreviated as avian influenza) are viral diseases of birds with high infectious and pathogenic effects. Once birds are diagnosed with infection, a large number of kills must be performed in order for Du Juehou to continue to infect other areas of birds, resulting in significant economic loss. Therefore, if a feed additive capable of improving fowl to resist fowl plague or avian influenza can be developed to improve fowl antibodies against fowl plague or avian influenza while feeding the feed, significant economic losses caused by infection can be avoided.

In addition, the porcine reproductive and respiratory syndrome is a high-infectivity, high-mortality and high-contact infectious disease, and has the characteristics of wide disease area, high transmission speed and the like. In addition, once mixed infection is carried out with the swine fever virus, the infection rate of the respiratory diseases of the piglet group is more remarkable, and the death rate of the piglets can be almost up to 80 percent under the condition of poor pig farm management. Therefore, once the pig farm has blue ear virus invasion and swine fever epidemic prevention work is sparse, the clustered infection of respiratory diseases is easy to be caused, and great economic loss is caused for the cultivated objects. Therefore, if a feed additive capable of improving the immunity of pigs to resist blue-ear disease and swine fever can be developed to feed sows and piglets, and antibodies of the pigs to resist blue-ear disease can be improved, the great economic loss caused by the infection of the viruses can be avoided.

Disclosure of Invention

The invention mainly aims to provide a feed additive and a feed containing the feed additive, which can enhance animal immunity and improve vaccine immunity effect, and can resist pathogenic bacteria infection and obesity. The feed additive of the invention comprises: a deactivated bacterial extract comprising a plurality of bacterial strain inclusions, wherein the bacterial strain inclusions comprise a polypeptide comprising: a translocation peptide for translocation; and a binding epitope. Wherein the translocation peptide may be located at the N-terminus of the polypeptide and the binding epitope may be located at the C-terminus of the polypeptide; vice versa.

In the feed additive of the present invention, the polypeptide may further optionally include: a linker polypeptide. Wherein the connecting polypeptide can be located between the translocation peptide and the binding epitope; or the translocation peptide is located between the linker polypeptide and the binding epitope. However, the present invention is not limited thereto; the relative positions of the translocation peptide, the binding epitope, and the linker polypeptide may be varied according to the intended purpose.

In the feed additive of the invention, the formula of the combined polypeptide can be an M cell target polypeptide or an intestinal epithelial target polypeptide. For example, the connecting polypeptide may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4.

In the feed additive of the invention, the translocation peptide is mainly derived from pseudomonas aeruginosa exotoxin. In one embodiment of the invention, the translocation peptide may comprise a Pseudomonas aeruginosa exotoxin A fragment with only functional region III removed. In another embodiment of the invention, the translocation peptide may comprise a Pseudomonas aeruginosa exotoxin A fragment that removes functional regions Ib and III. In yet another embodiment of the invention, the translocation peptide may comprise a fragment of Pseudomonas aeruginosa exotoxin A that removes functional regions II, ib and III. In a further embodiment of the invention, the translocation peptide may comprise a partial N-terminal amino acid sequence of a Pseudomonas aeruginosa exotoxin A fragment, e.g., as set forth in SEQ ID NO: 5.

In the feed additive of the invention, the binding epitope may be designed as desired against a particular disease. For example, the binding epitope may be a chicken newcastle disease (Newcastle disease, ND) virus epitope, an Avian Influenza (AI) virus epitope, a myostatin protein binding epitope, a porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus, PRRSV) epitope, or a swine fever (Class swine fever virus, CSFV) epitope.

In the feed additive of the present invention, the strain used is not particularly limited as long as it can express the polypeptide, and preferably a strain expressing the polypeptide in a large amount can be used in the feed additive of the present invention. For example, the strain may be an E.coli strain, a Bacillus subtilis strain, a Lactobacillus strain or an enterococcus strain.

The feed additive of the present invention may further comprise talc, ethylcellulose, resistant starch, etc. in addition to the aforementioned deactivated cell extract containing the polypeptide extract. Wherein the content of the deactivated thallus polypeptide extract can be 0.1-1 wt%, the content of talcum powder can be 67-87 wt%, and the content of ethyl cellulose can be 10.5-18 wt%; in order to enhance the sustained-release effect, resistant starch may be further added in an amount of 1.5 to 15 weight percent.

The preparation method of the feed additive of the invention can comprise the following steps: culturing the strain as described above; lysing the cultured strain and separating Inclusion bodies (Inclusion bodies), and selectively lysing the strain to obtain a polypeptide extract; the polypeptide extract is mixed with talcum powder and ethyl cellulose, and resistant starch and the like can be added, so that the feed additive can be obtained.

Furthermore, the present invention provides a feed comprising: a feed additive and a feed matrix as hereinbefore described. Wherein, the content of the feed additive can be 0.03 to 5 weight percent, and the content of the feed matrix can be 95 to 99.97 weight percent. Alternatively, the feed additive may be present in an amount of 0.05 to 1.5 weight percent and the feed matrix may be present in an amount of 98.5 to 99.95 weight percent. The feed substrate may be selected from feeds commonly used in the art as a feed substrate according to the animals to be fed (e.g., chickens, pigs, sheep, cattle, humans, etc.).

The feed additive of the invention can be mixed with a feed matrix to obtain the feed of the invention. When the feed provided by the invention is used for providing an animal, the vaccine injection titer can be effectively improved, the efficacy of the animal against specific diseases (such as chicken newcastle disease, chicken avian influenza or pig obesity, pig reproduction and respiration syndrome, swine fever and the like) is enhanced, and the immunity and productivity of the animal are further improved.

Drawings

FIGS. 1 and 2 are graphs showing the results of immunization against chicken Newcastle disease virus alone for chickens taking the feed containing the feed additive according to example 2 of the present invention;

FIG. 3 is a graph showing the results of the immunization of chickens fed with the feed additive of example 3 of the present invention against avian influenza virus alone.

Detailed Description

Other advantages and effects of the present invention will become apparent to those skilled in the art from the disclosure of the present specification, which is by way of specific examples. The invention is capable of other and different embodiments and its several details are capable of modifications and various other uses and applications, all of which are obvious from the description, without departing from the spirit of the invention.

Example 1 obesity

In this example, two fusion protein polypeptides are provided to elicit the production of myostatin protein binding epitope antibodies in an animal for blocking myostatin activity. The method of constructing the fusion protein polypeptide is generally as follows.

The DNA fragment of the myostatin binding epitope (Mt 4) was ligated into pP49 or pPE407 plastids (pET 23a and PE toxoid vector derivative plastids) cut with EcoR1 and XhoI restriction enzymes, respectively, such that the DNA fragment of the myostatin binding epitope was added to the C-terminus of the P49 or PE407 fragment. The plasmids containing the inserts were separately transfected into E.coli and strains were selected by ampicillin resistance. Once the sample is obtained, the machine will automatically generate a DNA sequence and display its results on a computer. Sequence analysis is performed to identify nucleotide sequences in nucleic acids or amino acids in polypeptides.

1-1: construction of P49L-Mt4 fusion protein polypeptide

A P49L-Mt4 fusion protein polypeptide comprising: translocation peptides, myostatin binding epitopes and linked polypeptides. Wherein the translocation peptide is derived from Pseudomonas aeruginosa exotoxin and is free of PE functional region II, ib and III fragments and is free of the organelle junction region PEIa, leaving only the N-terminal 49 amino acid (PE 49) fusion protein

Construction of pPE49 plastid

Pseudomonas aeruginosa exotoxin A (PE) polypeptides contain functional regions (domains) Ia, II, ib and III. A total of 613 amino acids was present, the full length PE-DNA fragment of which has been disclosed (Liao CW et al, applied Microbiology and Biotechnology, july 1995,Volume 43,Issue3,pp 498-507), and the sub-transferred strain was stored in Liao Lab. The PE functional region (domain) Ia portion of pET23a plastid containing PE-DNA and the functional regions II, ib and III-DNA fragments were deleted using genetic engineering techniques, leaving only the 49 amino acids (PE 49) at the N-terminus that did not contain the organelle junction PEIa, as shown in Table 1 below. Under the control of strong phage T7 transcription and (optionally) translation signals, the gene of interest is transfected into pET15b plastids; expression is induced by providing a T7 RNA polymerase in the host cell.

Table 1: translocation peptide PE49 sequence

A159 bp (base pair) DNA fragment encoding the nucleotide sequence of PE aa 1-49 was synthesized by PCR of PE-F1 and PE-R1 using the primers shown in Table 2 below, containing NdeI at the 5 '-end of the DNA and EcoR1 and XhoI at the 3' -end of the DNA. The 165bp PCR product was cut with XhoI and NdeI to isolate a 159bp fragment. Then, the resultant was transferred to a 5.9kb large DNA fragment obtained by cleaving pET15b with XhoI and NdeI to give a plasmid having EcoR1 and XhoI at the 3' -end of pPE-49 (6079 bp).

TABLE 2

Construction of pPE49 plastids containing a connecting polypeptide

linker-aDNA fragments were inserted into EcoR1 and Xho1 sites of pPE-49, as shown in Table 3 below. The DNA fragment and amino acid fragment sequences of the completed pPE-49-linker2 (pPE 49L) plastid are shown in Table 4 below. Wherein the amino acid fragment of pPE49L comprises: n-terminal translocation peptide and C-terminal junction polypeptide

Table 3: linker peptide Linker-a sequence

TABLE 4 Table 4

Mt4 gene design and plasmid construction of P49L-Mt4 containing Mt4

The myostatin protein binding epitope (Mt 4) is selected from a partial fragment of the myostatin binding region, and the DNA fragment and amino acid fragment sequences are shown in Table 5 below.

TABLE 5

The XhoI sites of pP49L plasmid were inserted into Sal I and XhoI sites excised from Mt4 synthetic DNA, as shown in Table 6 below. The completed pP49L-Mt4 plastid had the polypeptide sequences shown in Table 7 below. Finally, the plasmids containing the inserts were separately transformed into E.coli and strains were selected by ampicillin resistance.

TABLE 6

TABLE 7

1-2: construction of PE407-Mt4 fusion protein polypeptide

The PE407-Mt4 fusion protein polypeptide comprises: the translocation peptide and myostatin binding epitope. Wherein the translocation peptide is derived from Pseudomonas aeruginosa exotoxin, does not contain PE functional region Ib and III fragments, but contains organelle junction PE functional region Ia and II fragments.

Construction of pPE407 plastid

Under the control of strong phage T7 transcription and (optionally) translation signals, the PE407 target gene was transfected into pET15b plastids; expression is induced by providing a T7 RNA polymerase in the host cell. Plastid pPE407 was constructed as follows. A1224 bp DNA fragment encoding the nucleotide sequence of PE aa 1-407 was synthesized by PCR of PE-F2 and PE-R2 using the primers shown in Table 8 below. The 1238bp PCR product was cut with XhoI and NdeI to isolate a 1224bp fragment. Then the second was transferred into a 5.9kb large DNA fragment obtained by cleaving pET15b with XhoI and NdeI to yield plastid pPE-407 (7159 bp).

The nucleotide and amino acid sequences of the above PE fragments are polyhistidine (poly-His) epitopes flanked by linker sequences MGSSHHHHHH and LEHHHHHZ at the 5 '-and 3' - (or N-and C-) termini, respectively.

TABLE 8

Plasmid construction of PE407-Mt4 containing Mt4

The DNA fragment of myostatin protein binding epitope (Mt 4) (shown in Table 6) was ligated into pPE407 plastids (pET and PE toxoid vector derivative plastids) cut with EcoR1 and XhoI restriction enzymes, such that Mt4 was added to the C-terminus of the PE (. DELTA.III) fragment. The completed pPE407-Mt4 plastid had the polypeptide sequences as shown in Table 9 below. Finally, the plasmids containing the inserts were separately transformed into E.coli and strains were screened for resistance to ampicillin.

TABLE 9

1-3: mouse test-addition of PE407-Mt4 and P49L-Mt4 as feed additives

The PE407-Mt4 and P49L-Mt4 plastids obtained above were transformed into E.coli, respectively, and cultured. Then, the cultured E.coli is subjected to cell lysis and Inclusion body (Inclusion body) separation, and the strain is selectively lysed, thereby obtaining a polypeptide extract. The feed additive of the present invention was obtained by mixing 1% by weight of the polypeptide extract, 80% by weight of talc, 1% by weight of resistant starch and 18% by weight of ethylcellulose. 1.5 portions of feed additive and feed matrix: 98.5, and the feed used in the test of the present mice was obtained.

Male golden mice of 4 weeks of age were purchased, placed in one cage for each 4, and mice per cage were adjusted to a similar average weight in order to maintain the same environment for the groups subjected to different treatments in later experiments, and pellet feed and water were provided before the experiments to allow free feeding and isolation for one week. At the age of 5 weeks, the animal cages were randomly allocated as groups as shown in table 10 below, and 10g of each pellet feed and 10g of low fat (1%) feed sample and water were provided to allow free feeding, and after feeding for at least 1 week, 5-10g of each pellet feed and 5-10g of high fat (10%) feed sample and water were re-fed at the age of 6 weeks to allow free feeding, and feeding was performed for at least 4 weeks to 10 weeks.

Daily feed dosage and residual quantity were recorded during the experiment, and daily consumption was calculated. Mice were weighed weekly during the experiment and body weights at the beginning of the experiment were compared to those at the end of the experiment. Calculating the feed conversion ratio (Feed Conversion Rate, FCR) according to the formula: daily consumption = feed dosage-residual amount. And Anova statistical analysis was performed using Stat View software. The results are shown in Table 11 below.

Three mice were orally fed with a feed additive containing PE407-Mt4 and P49L-Mt4 polypeptides, and blood was collected from the eye sockets by puncturing the rear plexus at weeks 6, 8, and 10. The potency of specific antibodies against Mt4 antigen was detected from serum. Blood samples were collected and the potency of anti-Mt 4 specific antibodies was detected in ELISA using serial 10-fold dilutions. The results are shown in Table 12 below.

Table 10

TABLE 11

Table 12: mt 4-specific serum IgG activity was detected in serial dilutions (1:200-1:1250) by ELISA assays in different serum samples.

The foregoing results demonstrate that when PE407-Mt4 and P49L-Mt4 are used as myostatin polypeptide feed additives for animals, including PE407-Mt4 and P49L-Mt4 comprising Mt epitopes have an induction effect of myostatin protein binding epitope specific antibodies. Therefore, the recombinant myostatin can induce immune response to myostatin through a feed additive way, so that the weight of the golden mice is increased, and the feed conversion rate is enhanced. This is an important step in transforming cells into food vaccines to improve meat production and combat muscle wasting genetic disease in farm animals (Zhang T et al, BMC Biotechnol.2012Dec 19;12:97;Aravind S et al, J Virol methods.2012nov;185 (2): 234-8).

In addition, the feed additive of this example was added to a feed for medium or large pigs at a ratio of 0.05% or 0.025%, and a physical test of a pork pig was performed. The test results confirm that the middle pigs and the large pigs of the ternary pig breeds take the feed containing the feed additive of the embodiment, the convex abdominal umbilical line disappears after one month, the body beautifying pigs with double ridges and double strands can be seen, the pork quality after the carcasses is excellent, the intramuscular fat is fine, and poor water-like meat is not presented any more. Therefore, the feed additive of the embodiment can replace clenbuterol, and is a safe and environment-friendly product.

EXAMPLE 2 enhancing the immunity of chickens against newcastle disease (fowl plague) alone

In the embodiment, a fusion protein polypeptide feed additive is provided, and the immune efficacy of chicken newcastle disease vaccine injection can be enhanced by adding the fusion protein polypeptide feed additive into feed. The construction method of the fusion protein polypeptide is approximately as follows.

TABLE 13

Plasmid construction of PE425-NDV-1 containing NDV-1

A DNA fragment of the Th1 epitope of newcastle disease virus NDV (NDV-1) (as shown in Table 13) was ligated into pPE425 plastids (pET and PE toxoid vector derivative plastids) cut with EcoR1 and XhoI restriction enzymes, such that NDV-1 was added to the C-terminus of the PE (. DELTA.III) fragment. The completed pPE425-NDV-1 plastid has the polypeptide sequences shown in Table 14 below. Finally, the plasmids containing the inserts were separately transformed into E.coli and strains were selected by ampicillin resistance.

TABLE 14

Chicken test for enhancing immunity

The plastids obtained above were transferred into E.coli, respectively, and cultured. Then, the cultured E.coli is subjected to cell lysis and Inclusion bodies (Inclusion bodies) are separated out, and a polypeptide extract is obtained after washing. The extracted polypeptide extract is 1 weight percent, mixed with the talcum powder which is 81 weight percent and the ethyl cellulose which is 18 weight percent and other extenders, and then crushed and ground by a super-powder machine, thus obtaining the feed additive of the invention.

300 commercial hens with similar weight and good health and 1 day old were randomly divided into an antibiotic group (0.03 w/w% Colistin and 0.03w/w% Enramycin were added), a control group (no antibiotic and feed additive were added), and a feed additive group (low dose 0.025w/w%, medium dose 0.05w/w%, high dose 0.1w/w% feed additive) with 4 replicates per treatment group, and 15 replicates per treatment group. The feed additive and antibiotics are respectively added into the feed at 0-4 weeks and 10-13 weeks, and the weight gain and feed intake of the 0-15 week old laying hens are observed and the feed efficiency is calculated. The egg laying rate, egg weight, egg yield and feed egg replacement rate of 19-26 week old chickens were measured, and the antibody titer of the serum was measured at 6, 16 and 26 weeks. In addition, chickens were kept in flat fields for 0-16 weeks and transferred to egg cages for 16 weeks, and all chickens were kept in 6-15 weeks with 0.05% coccidiosis (Decoguinate and Lasalocid) added to the diet, respectively. The results are shown in FIGS. 1 and 2.

As shown in the results of FIG. 1 and FIG. 2, the feed containing the feed additive of the embodiment can significantly enhance the immune efficacy (p < 0.01) of chicken newcastle disease virus when taken by chickens of 0-4 weeks old and chickens of 10-13 weeks old. In addition, the feed containing the feed additive of the embodiment has no side effect on the laying hens, and has no obvious differences in laying rate, egg weight, egg yield and feed egg replacement rate compared with the control group.

Example 3-enhancing immunity of chickens against avian influenza alone

In this example, a fusion protein polypeptide is provided that enhances the antibody titer of chicken against avian influenza M2 antigen alone. The construction method of the fusion protein polypeptide is approximately as follows.

TABLE 15

A DNA fragment of the Th1 epitope (M2-Nr 7) of avian influenza virus (as shown in Table 15) was ligated into pPE425 plastids (pET and PE toxoid vector derivative plastids) cut with EcoR1 and Sal 1 restriction enzymes, such that M2-Nr7 was added to the C-terminus of the PE (. DELTA.III) fragment. The completed pPE425-M2-Nr7 plastids had the polypeptide sequences as shown in Table 16 below. Finally, the plasmids containing the inserts were separately transformed into E.coli and strains were selected by ampicillin resistance.

Table 16

Chicken test for enhancing immunity

The chicken of this example was tested the same as example 2, with the only difference that: the feed additive added to the feed for chickens of this example contained a polypeptide additive produced by E.coli transformed with the fusion protein polypeptide implant of this example. The results are shown in FIG. 3.

As shown in the results of FIG. 3, the feed added with 0.1w/w% of the feed additive of the embodiment can enhance the antibody titer of the avian influenza surface antigen M2 and has a significant difference (P < 0.01) relative to the antibody titer of the control group when the feed additive is taken by 0-4-week-old chickens and 10-13-week-old chickens. As for the group of feeds added with 0.05w/w% of the feed additive of this example, there was also a significant difference (P < 0.05) with respect to the antibody force value of the control group. The group of feeds with 0.025w/w% of the feed additive of this example had a tendency to differ (P < 0.1) relative to the antibody titers of the control group. In addition, the feed containing the feed additive of the embodiment is taken, and compared with the control group, the feed additive has no obvious difference in laying rate, egg weight, egg yield and feed egg change rate.

Pig test for enhancing immunity

EXAMPLE 4 enhancing immunity against porcine reproductive and respiratory syndrome and swine fever

In this example, two fusion protein polypeptides are provided to induce immunity in pigs to enhance resistance to porcine reproductive and respiratory syndrome and swine fever. The construction method of the two fusion protein polypeptides is approximately as follows.

Construction method and sequence of fusion protein polypeptide SEQ ID NO. 28 and SEQ ID NO. 29

TABLE 17

TABLE 18

DNA fragments of Th1 epitope PR17 of blue-ear virus and Th1 epitope SF1 of swine fever (as shown in tables 17 and 18) were ligated into pPE425 plastids (pET and PE toxoid vector derivative plastids) cut with EcoR1 and Xho1 restriction enzymes, such that PR17 and SF1 were added at the C-terminus of the PE (. DELTA.III) fragment. The completed pPE 425-PR17 and pPE425-CF1 plastids had the polypeptide sequences shown in Table 19 and Table 20 below. Finally, the plasmids containing the inserts were separately transformed into E.coli and strains were selected by ampicillin resistance.

TABLE 19

Table 20

Pig test method

Arrangement of pregnant sow (binary white pig) test

The test adopts a single factor experimental design, the number of the selected fetuses is second or third, 27 sows which are normally pregnant and are healthy and pregnant for 100 days are randomly divided into three treatment groups (two test groups A.5, A.10 and a control group B), each treatment group is repeated three times, and each repetition is three sows. Each treatment group is fed separately, and the live blue-ear virus vaccine and the swine fever vaccine of the sow are administered with a dose before mating, and the two vaccines are not administered any more during pregnancy, and other immunization programs and daily care follow the daily management method of a pig farm.

The PE425-PR17 and PE425-SF1 plastid prepared in the above way are respectively transferred into Escherichia coli. Then, the cultured E.coli is subjected to cell lysis and Inclusion body (Inclusion body) separation to obtain a polypeptide extract. And selectively lysing the strain to obtain the polypeptide extract. Two polypeptide extracts (PE 425-PR17 and PE425-SF 1) are added into the feed additive invented by the test group, wherein the addition content of the two polypeptide extracts is 1 percent by weight, the content of talcum powder is 81 percent by weight, and the content of ethyl cellulose is 18 percent by weight, so that the feed additive YYP is obtained. The feed additive YYP was mixed with a feed base (as shown in table 21 below) at 0.05%:99.95% by weight of the feed composition of the test group A.5 was obtained. The feed additive and the feed matrix are mixed according to the ratio of 0.1 percent: 99.9% by weight of the feed combination of the test group A.10 was obtained. The above formulation was stirred well and fed twice daily (once in the morning and evening) for three treatments per test group. The sow starts to feed for hundreds of days during pregnancy until the piglet weans and starts to end the test after slaughtering (about 32 days). The control group was not treated identically except that the additive was not taken. The blood collection is carried out on all sows in the stage that the sows are not taken yet (99 days of pregnancy), the second blood collection is carried out on the first day after the birth of the suckling pigs, the third blood collection is carried out on the 3 days before weaning, and the blood collection interval time is about 15 days.

Table 21: basic ration composition and nutrition level of sow feed in later pregnancy and lactation period

* The premix comprises Cu 12.6mg,Fe 135.3mg,Zn 98.3mg,Mn 44.5mg,Co 1.1mg,Se 0.7mg,I 0.6mg, vitamin A16000 IU, vitamin D34550 IU, vitamin E53.5 IU, vitamin K4.1 mg, vitamin B2.7 mg, vitamin B3 41.50mg, vitamin B6.50 mg, vitamin B12.04 mg, folic acid 6.73mg, biotin 0.36mg and choline 0.34mg.

Pregnant sow (binary white pig) test results

Compared with the control group, the polypeptide feed additive YYP is added into daily ration in the aspect of sow productivity, and has no statistically significant difference (P > 0.05) in the influence on the total number of farrowing of the sow, the number of weaned pigs and the survival rate of the weaned pigs. However, the addition of the polypeptide feed additive YYP (0.05% and%) has an effect of improving the feed intake of the sow (P < 0.05), the weaning-oestrus interval of the sow is shortened, and the ratio of the diarrhea of the suckling piglet is reduced, so that the efficiency of improving the feed intake of the sow (P < 0.05) is statistically significant.

Compared with the control group, after a certain amount of polypeptide feed additive YYP is added into the ration of the sow, the anti-porcine reproductive and respiratory syndrome virus antibody value in serum is detected, so that the IgG level of the sow antibody can be enhanced to a certain extent, and the S/P value (P < 0.05) of the very high level porcine reproductive and respiratory syndrome virus antibody IgG is maintained. Comparing the control group, both test groups A.5 and A.10 were effective in reducing the anti-physical value dispersion of the group, resulting in a percent antibody positive conversion (P < 0.05).

Table 22: influence of polypeptide additives on anti-physical value (S/P) and yang conversion rate of blue-ear viruses in sow serum

* Data for a.5 and a.10 compared to control, the T-test statistics reached significant difference levels (P < 0.05).

Detecting the swine fever antibody titers in the serum thereof delays the decline of the swine fever virus antibody IgG level of the sow to a certain extent, and simultaneously maintains the positive rate of the high-level swine fever virus antibody IgG (P < 0.05), but does not affect the IgA content of the swine fever virus antibody in the sow serum (P > 0.05).

Table 23: influence of polypeptide additive on swine fever physical strength and yang conversion rate in sow serum

Arrangement of piglet experiments

The experiment adopts a single factor experiment design, 60 piglets are selected, the number of the piglets is similar, the variety is consistent (binary white pigs), the male and female pigs are half, the weight of the piglets is similar, weaned piglets are 21 days old, the piglets are randomly divided into three treatments (a.0.5, b.1 and control group c), each treatment is repeated for 4 times, and 5 piglets are repeated every time. The immunization program of the piglets, the live porcine reproductive and respiratory syndrome vaccine and the swine fever vaccine are fed to each component column, the first needle is applied at the age of 20 days, the second porcine reproductive and respiratory syndrome vaccine is applied at the age of 55 days, the blue reproductive and respiratory syndrome vaccine is not applied, and other vaccine control and daily nursing follow the daily management method of a pig farm.

The PE425-PR17 and PE425-SF1 plastid prepared in the above way are respectively transferred into Escherichia coli. Then, the cultured E.coli is subjected to cell lysis and Inclusion body (Inclusion body) separation to obtain a polypeptide extract. And selectively lysing the strain to obtain the polypeptide extract. Two polypeptide extracts (PE 425-PR17 and PE425-SF 1) with the addition content of 1% by weight, the talcum powder with the content of 79% by weight and the ethylcellulose with the content of 18% by weight are added into the feed additive disclosed by the test group to obtain the feed additive YYP. The feed additive YYP was mixed with a feed base (as shown in table 24 below) at 0.05%:99.9% by weight of the feed combination of test group a.0.5 was obtained. Feed additive YYP and feed matrix in 0.1%:99.9% by weight of the feed composition of the test group b.1 was obtained. The above formulation was stirred uniformly, 4 treatments per test group. When the test is carried out, the feed is added into the feed tower, and once the residual quantity in the feed barrel is found to be small, the feed can be added again, so that the feed for piglets can be maintained at any time.

Table 24: piglet breeding feed formula

Piglet test results

The piglet feed intake and the weight gain effect (P < 0.05) of the piglet can be obviously improved by adding a certain amount of polypeptide feed additive (0.05% or 0.1%) YYP into the ration. Does not affect the feed conversion ratio (P > 0.05) of the weaned pigs, and does not promote the occurrence and aggravation of cough and asthma, diarrhea and death of the weaned pigs (P > 0.05).

In general, for effective swine fever control of piglets, swine fever vaccines must be given by two immunization injections (e.g., three weeks and six or eight weeks of age), and the protection efficiency is not sufficient after the first injection. In a piglet test after 20-day-old piglets are given by injecting swine fever vaccine, the serum positive reaction of 45-day-old piglets in a control group is still poor, and the serum neutralization power of 1:16 cannot be achieved. As shown in Table 25 below, the serum blocking rate of piglets in the CSFV IDEXX kit was less than 30% and the total number of piglets in the 45-day-old control group was as high as 6/16, whereas the blocking rate was >40% and was only 7/16. The serum of the control group only reaches 40% coverage after two weeks of immune injection for 4.4 piglets, and the serum of other 5.6 piglets does not reach 40% (40% coverage cannot be reached and is regarded as negative reaction).

As shown in Table 25 below, the serum blocking rate of the 45-day-old piglets of test group a.0.5 was 2/16 of the total number of pigs at 30% or less. The serum blocking rate of the test group b.1 piglets at 45 days age is also 2/16 of the total pig count compared with the pig count below 30%, while the blocking rate of the test group a.1 piglets at 40% is as high as 14/16.

In addition, control group c had not been given a second swine fever vaccine stage at 45 days of age, and the 40% coverage rate positive rate was only 44%, indicating that the herd had no protection against swine fever disease. When the protective force is detected based on the 50% blocking rate, it is known that the test group a.0.5 has no protective force for up to 62.5%, but the test group b.1 for 45-day-old piglets has protective force against swine fever diseases when the positive rate of the 50% blocking rate is up to 87.5%.

By statistically analyzing the serum antibody detection results of the test pigs b.1 formulated with the polypeptide feed additive YYP (0.1%), it was found that the pig group had very significant improvement in swine fever antibody blocking values (p=0.004, P < 0.05) for piglets, whereas the test pigs a.0.5 formulated with the polypeptide feed additive YYP (0.05%) slightly reached the level of difference (p=0.053, P < 0.1).

Table 25: influence of polypeptide additive YYP on swine fever physical strength resistance and yang conversion rate in piglet serum

Judgment standard: a.0.5 p=0.053, b.1 p=0.004 (t.test)

Hog cholera antibody (blocking value): the antibody detection adopts ELISA method, and after vaccine immunization, the blocking rate of the antibody is more than or equal to 40%, which indicates that the antibody is positive; the blocking rate is less than or equal to 30 percent, which indicates that the antibody is negative; the blocking rate is between 30 and 40% and the animals should be retested after several days. The blocking rate of the swine fever antibody of the swinery reaches 100 percent or reaches 85 percent by >50 percent, and the dispersion is less than 25 percent, which indicates that the immune condition of the swinery is good; dispersion between 25% and 40% indicates a general immune effect. The blocking rate of the swine fever antibody of the sow is more than 50 percent, which indicates that the immunization is qualified. IDEXX kit.

In addition, the current field virulent strains are very complex, and the effectiveness of blue-ear vaccines is difficult to evaluate. In a common pig farm, the blue-ear disease resistance of piglets is low before 0 to 5 weeks of age, the positive conversion rate is still low, and the group force rate dispersion rate of piglets is high, so that the piglet is a common disease. Once the pig farm or the surrounding has the blue-ear disease epidemic situation, the piglet group in the empty window period is most easily affected by the blue-ear virus, so that the fast epidemic situation is spread and is not prevented.

As shown in the results in table 26 below, the number of pigs with serum S/P values of 0.4 or less at 45 days of age of the piglets in the test group b.1 was 0%, the serum positive transfer rate was 100%, and the serum response in the test group was statistically significantly different from that in the control group c (P < 0.05) as shown by the average value, standard deviation and p=0.04 of T-test.

The number of pigs with the S/P value of the serum of the 45-day-old piglets of the test group a.0.5 below 0.4 is 30% compared with the total number of pigs, and the serum positive transfer rate is only 70%. The number of pigs of the control group c with the serum S/P value below 0.4 at the age of 45 days is 40% compared with the total number of pigs, which shows that the two groups of pigs still have low immunity at the age of 45 days and do not have the capability of resisting blue ear diseases.

Table 26: YYP influence of additive on physical strength and sexual valence resistance and yang transformation rate of porcine reproductive and respiratory syndrome in piglet serum

Judgment standard: a.0.5 p=0.33, b.1 p=0.04 (t.test)

The antibody detection adopts a PRRS-ELISA method of IDEXX kit, and after vaccine immunization, the S/P value of the antibody reaches more than 0.4, which indicates that the antibody is positive; 0.4 or less, which indicates antibody negativity. The piglet group with the dispersion of S/P value below 50% after vaccine administration has the collective immune response of the vaccine. When the dispersion is too large, the virus invasion phenomenon is indicated.

In summary, after the live vaccine against blue-ear of piglet and the vaccine against swine fever are administered at 20 days old, the experimental group b.1 with 0.1% of the polypeptide feed additive YYP added in the daily ration is compared with the control group, and it is known that the experimental group b.1 has the advantages of enhancing the immune efficiency of the two vaccines, and remarkably improving the antibody titer level and the positive rate (P < 0.05).

Pig farm field test

After the pregnant sow and the piglet are tested, a certain amount of YYP products are added into the basic ration of the sow and the weaned piglet, so that the production benefits of the sow and the weaned piglet can be improved to a certain extent. Thus, YYP 0.05.05% was added to the sow feed (stock and nursing feed), and YYP 0.1.1% was added to the creep feed and nursing feed for piglets. And (3) performing an expanded field test in a pig farm of thousands of sows, and observing the benefit of the additive on a blue-ear disease infection field.

The test site is selected in a large pig farm in boro county, guangdong province of China, and the blue ear disease of the pig farm is a common disease and is annoyed for several years. After 2017/1/10 of the vaccine against live blue-ear disease, abortion in the birthing house and mortality of piglets were not improved. Therefore, starting at 2017/1/15, YYP 0.05.05% was added to the sow feed (mother and baby feed) and YYP 0.1.1% was added to the creep feed and the nursery feed for piglets. Since YYP feed additives are not a mechanism of action of drugs, and the infection mechanism of blue-ear disease is very complex, the transmission pipeline is also versatile, so long-term observation is required to be effective. The efficacy of YYP for blocking reproductive disorders was judged by analysis of the following Table 27, evaluation of the benefit of YYP feed additives in such pig farms, observation of the weak litter productivity of sow production in the pig farm over four months (2017/1-4). And the respiratory diseases of the nursery pigs in the nursery house after the five months are obviously improved.

Table 27

As shown in the results of table 27, when the test pigs were fed with the feed added with the feed additive of this example, the rate of occurrence of the blue ear disease and weaning was greatly reduced.

In summary, when the feed added with the feed additive of the invention is fed to animals, the effect of the animals against specific diseases (such as chicken newcastle disease, avian influenza, obesity or pig reproduction and respiration syndrome) can be effectively improved, and the productivity of the animals can be further improved.

The above embodiments are merely illustrative, and the claimed invention should not be limited to the above embodiments, but rather should be construed according to the claims.

Sequence listing

<110> Liao Tingzhang

<120> feed additive and feed comprising the same

<130> A1115

<140> 201811054003.8

<141> 2018-09-10

<160> 29

<170> SIPOSequenceListing 1.0

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Ser Phe His Gln Leu Pro Ala Arg Ser Pro Ala Pro Leu Gln

1 5 10

<210> 2

<211> 22

<212> PRT

<213> Artificial Sequence

<220>

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Ser Ser Phe His Leu Phe His His Leu Pro Ala Arg Ala Pro Leu Ala

1 5 10 15

Pro Ser Glu Leu Gln Pro

20

<210> 3

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesized peptide

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Ser Thr Pro Phe His Pro Leu Pro Ala Arg Lys Pro Leu Pro Leu Gln

1 5 10 15

Pro

<210> 4

<211> 212

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<220>

<223> Synthesized peptide

<400> 4

Glu Phe His Met Val Asp Gly Met Ser Ile Arg Ala Lys Arg Arg Lys

1 5 10 15

Arg Ala Ser Ala Thr Gln Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr

20 25 30

Cys Pro Pro Asp Ile Ile Pro Lys Val Glu Gly Lys Thr Ile Ala Glu

35 40 45

Gln Ile Leu Gln Tyr Gly Ser Met Gly Val Phe Phe Gly Gly Leu Gly

50 55 60

Ile Gly Thr Gly Ser Gly Thr Gly Gly Arg Thr Gly Tyr Ile Pro Leu

65 70 75 80

Gly Thr Arg Pro Pro Thr Ala Thr Asp Thr Leu Ala Pro Val Arg Pro

85 90 95

Pro Leu Thr Val Asp Pro Val Gly Pro Ser Asp Pro Ser Ile Val Ser

100 105 110

Leu Val Glu Glu Thr Ser Phe Ile Asp Ala Gly Ala Pro Thr Ser Val

115 120 125

Pro Ser Ile Pro Pro Asp Val Ser Gly Phe Ser Ile Thr Thr Ser Thr

130 135 140

Asp Thr Thr Pro Ala Ile Leu Asp Ile Asn Asn Asn Thr Val Thr Thr

145 150 155 160

Val Thr Thr His Asn Asn Pro Thr Phe Thr Asp Pro Ser Val Leu Gln

165 170 175

Pro Pro Thr Pro Ala Glu Thr Gly Gly His Phe Thr Leu Ser Ser Ser

180 185 190

Thr Ile Ser Thr His Asn Tyr Glu Glu Ile Pro Met Asp Thr Lys Asp

195 200 205

Glu Leu Leu Glu

210

<210> 5

<211> 49

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesized peptide

<400> 5

Ala Glu Gln Leu Val Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys Val

1 5 10 15

Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp Pro

20 25 30

Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu His Tyr Cys Met Glu

35 40 45

Phe

<210> 6

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthesized primer

<400> 6

ccccatatgg ccgaagaagc t 21

<210> 7

<211> 49

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthesized primer

<400> 7

tttctcgagt tgaattccat ggagtagttc atcactccct ggccgttgg 49

<210> 8

<211> 78

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthesized DNA

<400> 8

gaattcagca gctttcatct gttccaccat ctgccagcgc gtgcgccatt agcgccttct 60

gaattacagc ccctcgag 80

<210> 9

<211> 284

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthesized DNA

<400> 9

gtttaacttt aagaaggaga tataccatgg ccgaacaatt ggtggacctc tggaacgaat 60

gcgccaaagc ctgcgtgctc gacctcaagg acggcgtgcg ttccagccgc atgagcgtcg 120

acccggccat cgccgacacc aacggccagg gcgtgctgca ctactgcatg gaattcagca 180

gctttcatct gttccaccat ctgccagcgc gtgcgccatt agcgccttct gaattacagc 240

ccctcgagca ccaccaccac caccactgag atccggctgc taac 292

<210> 10

<211> 80

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesized peptide

<400> 10

Met Ala Glu Gln Leu Val Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys

1 5 10 15

Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp

20 25 30

Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu His Tyr Cys Met

35 40 45

Glu Phe Ser Ser Phe His Leu Phe His His Leu Pro Ala Arg Ala Pro

50 55 60

Leu Ala Pro Ser Glu Leu Gln Pro Leu Glu His His His His His His

65 70 75 80

<210> 11

<211> 252

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthesized DNA

<400> 11

gaattcgtcg acgtgttttt acaaaaatat cctcatacgc acctggtcca tcaggcgctc 60

gacgtgtttt tacaaaaata tcctcatacg cacctggtcc atcaggcgct cgacgtgttt 120

ttacaaaaat atcctcatac gcacctggtc catcaggcgc tcgacgtgtt tttacaaaaa 180

tatcctcata cgcacctggt ccatcaggcg ctcgagcacc accaccacca ccactgagat 240

ccggctgcta ac 260

<210> 12

<211> 78

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesized peptide

<400> 12

Glu Phe Val Asp Val Phe Leu Gln Lys Tyr Pro His Thr His Leu Val

1 5 10 15

His Gln Ala Leu Asp Val Phe Leu Gln Lys Tyr Pro His Thr His Leu

20 25 30

Val His Gln Ala Leu Asp Val Phe Leu Gln Lys Tyr Pro His Thr His

35 40 45

Leu Val His Gln Ala Leu Asp Val Phe Leu Gln Lys Tyr Pro His Thr

50 55 60

His Leu Val His Gln Ala Leu Glu His His His His His His

65 70 75

<210> 13

<211> 488

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthesized DNA

<400> 13

gtttaacttt aagaaggaga tataccatgg ccgaacaatt ggtggacctc tggaacgaat 60

gcgccaaagc ctgcgtgctc gacctcaagg acggcgtgcg ttccagccgc atgagcgtcg 120

acccggccat cgccgacacc aacggccagg gcgtgctgca ctactgcatg gaattcagca 180

gctttcatct gttccaccat ctgccagcgc gtgcgccatt agcgccttct gaattacagc 240

ccctcgacgt gtttttacaa aaatatcctc atacgcacct ggtccatcag gcgctcgacg 300

tgtttttaca aaaatatcct catacgcacc tggtccatca ggcgctcgac gtgtttttac 360

aaaaatatcc tcatacgcac ctggtccatc aggcgctcga cgtgttttta caaaaatatc 420

ctcatacgca cctggtccat caggcgctcg agcaccacca ccaccaccac tgagatccgg 480

ctgctaac 504

<210> 14

<211> 148

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesized peptide

<400> 14

Met Ala Glu Gln Leu Val Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys

1 5 10 15

Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp

20 25 30

Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu His Tyr Cys Met

35 40 45

Glu Phe Ser Ser Phe His Leu Phe His His Leu Pro Ala Arg Ala Pro

50 55 60

Leu Ala Pro Ser Glu Leu Gln Pro Leu Asp Val Phe Leu Gln Lys Tyr

65 70 75 80

Pro His Thr His Leu Val His Gln Ala Leu Asp Val Phe Leu Gln Lys

85 90 95

Tyr Pro His Thr His Leu Val His Gln Ala Leu Asp Val Phe Leu Gln

100 105 110

Lys Tyr Pro His Thr His Leu Val His Gln Ala Leu Asp Val Phe Leu

115 120 125

Gln Lys Tyr Pro His Thr His Leu Val His Gln Ala Leu Glu His His

130 135 140

His His His His

145

<210> 15

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthesized primer

<400> 15

ccccatatgg ccgaagaagc t 21

<210> 16

<211> 44

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthesized primer

<400> 16

tttctcgagg aattcgacgt cgccgccgtc gccgaggaac tccg 44

<210> 17

<211> 504

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesized peptide

<400> 17

Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro

1 5 10 15

Arg Gly Ser His Met Ala Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys

20 25 30

Ala Lys Ala Cys Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg

35 40 45

Met Ser Val Asp Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu

50 55 60

His Tyr Ser Met Val Leu Glu Gly Gly Asn Asp Ala Leu Lys Leu Ala

65 70 75 80

Ile Asp Asn Ala Leu Ser Ile Thr Ser Asp Gly Leu Thr Ile Arg Leu

85 90 95

Glu Gly Gly Val Glu Pro Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg

100 105 110

Gln Ala Arg Gly Ser Trp Ser Leu Asn Trp Leu Val Pro Ile Gly His

115 120 125

Glu Lys Pro Ser Asn Ile Lys Val Phe Ile His Glu Leu Asn Ala Gly

130 135 140

Asn Gln Leu Ser His Met Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp

145 150 155 160

Glu Leu Leu Ala Lys Leu Ala Arg Asp Ala Thr Phe Phe Val Arg Ala

165 170 175

His Glu Ser Asn Glu Met Gln Pro Thr Leu Ala Ile Ser His Ala Gly

180 185 190

Val Ser Val Val Met Ala Gln Thr Gln Pro Arg Arg Glu Lys Arg Trp

195 200 205

Ser Glu Trp Ala Ser Gly Lys Val Leu Cys Leu Leu Asp Pro Leu Asp

210 215 220

Gly Val Tyr Asn Tyr Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp Thr

225 230 235 240

Trp Glu Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys His

245 250 255

Asp Leu Asp Ile Lys Pro Thr Val Ile Ser His Arg Leu His Phe Pro

260 265 270

Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His Leu

275 280 285

Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln

290 295 300

Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu

305 310 315 320

Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala

325 330 335

Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu

340 345 350

Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser

355 360 365

Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala

370 375 380

Asn Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly Glu Cys

385 390 395 400

Ala Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro

405 410 415

Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Glu Phe Val Phe

420 425 430

Leu Gln Lys Tyr Pro His Thr His Leu Val His Gln Ala Leu Asp Val

435 440 445

Phe Leu Gln Lys Tyr Pro His Thr His Leu Val His Gln Ala Leu Asp

450 455 460

Val Phe Leu Gln Lys Tyr Pro His Thr His Leu Val His Gln Ala Leu

465 470 475 480

Asp Val Phe Leu Gln Lys Tyr Pro His Thr His Leu Val His Gln Ala

485 490 495

Leu Glu His His His His His His

500

<210> 18

<211> 213

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthesized DNA

<400> 18

gaattccttt tattgctccg cgatgaacta aaaaactatg gtgaagcggt gtctctgatt 60

cttttattga atacttcagc ctgtatgtat agtaaggacg agctcctact ggttgcagtt 120

ggaaaaatgc aacagtttcg tgacgagctt aagttattgc tctacctaac agaactaaca 180

acggtcttcc gcgatgaact gaaactactc gag 213

<210> 19

<211> 71

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesized peptide

<400> 19

Glu Phe Leu Leu Leu Leu Arg Asp Glu Leu Lys Asn Tyr Gly Glu Ala

1 5 10 15

Val Ser Leu Ile Leu Leu Leu Asn Thr Ser Ala Cys Met Tyr Ser Lys

20 25 30

Asp Glu Leu Leu Leu Val Ala Val Gly Lys Met Gln Gln Phe Arg Asp

35 40 45

Glu Leu Lys Leu Leu Leu Tyr Leu Thr Glu Leu Thr Thr Val Phe Arg

50 55 60

Asp Glu Leu Lys Leu Leu Glu

65 70

<210> 20

<211> 523

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesized peptide

<400> 20

Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro

1 5 10 15

Arg Gly Ser His Met Ala Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys

20 25 30

Ala Lys Ala Cys Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg

35 40 45

Met Ser Val Asp Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu

50 55 60

His Tyr Ser Met Val Leu Glu Gly Gly Asn Asp Ala Leu Lys Leu Ala

65 70 75 80

Ile Asp Asn Ala Leu Ser Ile Thr Ser Asp Gly Leu Thr Ile Arg Leu

85 90 95

Glu Gly Gly Val Glu Pro Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg

100 105 110

Gln Ala Arg Gly Ser Trp Ser Leu Asn Trp Leu Val Pro Ile Gly His

115 120 125

Glu Lys Pro Ser Asn Ile Lys Val Phe Ile His Glu Leu Asn Ala Gly

130 135 140

Asn Gln Leu Ser His Met Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp

145 150 155 160

Glu Leu Leu Ala Lys Leu Ala Arg Asp Ala Thr Phe Phe Val Arg Ala

165 170 175

His Glu Ser Asn Glu Met Gln Pro Thr Leu Ala Ile Ser His Ala Gly

180 185 190

Val Ser Val Val Met Ala Gln Thr Gln Pro Arg Arg Glu Lys Arg Trp

195 200 205

Ser Glu Trp Ala Ser Gly Lys Val Leu Cys Leu Leu Asp Pro Leu Asp

210 215 220

Gly Val Tyr Asn Tyr Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp Thr

225 230 235 240

Trp Glu Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys His

245 250 255

Asp Leu Asp Ile Lys Pro Thr Val Ile Ser His Arg Leu His Phe Pro

260 265 270

Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His Leu

275 280 285

Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln

290 295 300

Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu

305 310 315 320

Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala

325 330 335

Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu

340 345 350

Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser

355 360 365

Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala

370 375 380

Asn Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly Glu Cys

385 390 395 400

Ala Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro

405 410 415

Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser Thr

420 425 430

Arg Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala Glu Phe

435 440 445

Leu Leu Leu Leu Arg Asp Glu Leu Lys Asn Tyr Gly Glu Ala Val Ser

450 455 460

Leu Ile Leu Leu Leu Asn Thr Ser Ala Cys Met Tyr Ser Lys Asp Glu

465 470 475 480

Leu Leu Leu Val Ala Val Gly Lys Met Gln Gln Phe Arg Asp Glu Leu

485 490 495

Lys Leu Leu Leu Tyr Leu Thr Glu Leu Thr Thr Val Phe Arg Asp Glu

500 505 510

Leu Lys Leu Leu Glu His His His His His His

515 520

<210> 21

<211> 360

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthesized DNA

<400> 21

gaattccttt tagaattgct cgaggataaa ctaactgaag tggaaacacc tacaattcgc 60

aacgagtggc tgaaacttga ggaccgtctt acggaagtgg aaacccctat ccgcaatgag 120

tggttattat tgaccgaggt tgaaactcca atacgtaacg aatggttgct cctcactgag 180

gtcgagacac cgacaattcg caacgaatgg ctaaagctag aagatcgcct gacggaggta 240

gagacgccta ttcgtaatga atggctgctt cttaccgaag tggaaacccc tatccgtaat 300

gagtggcttt tattaactga ggttgagact ccaacaatcc gcaacgaatg gtgagtcgac 370

<210> 22

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesized peptide

<400> 22

Glu Phe Leu Leu Glu Leu Leu Glu Asp Lys Leu Thr Glu Val Glu Thr

1 5 10 15

Pro Thr Ile Arg Asn Glu Trp Leu Lys Leu Glu Asp Arg Leu Thr Glu

20 25 30

Val Glu Thr Pro Ile Arg Asn Glu Trp Leu Leu Leu Thr Glu Val Glu

35 40 45

Thr Pro Ile Arg Asn Glu Trp Leu Leu Leu Thr Glu Val Glu Thr Pro

50 55 60

Thr Ile Arg Asn Glu Trp Leu Lys Leu Glu Asp Arg Leu Thr Glu Val

65 70 75 80

Glu Thr Pro Ile Arg Asn Glu Trp Leu Leu Leu Thr Glu Val Glu Thr

85 90 95

Pro Ile Arg Asn Glu Trp Leu Leu Leu Thr Glu Val Glu Thr Pro Thr

100 105 110

Ile Arg Asn Glu Trp Val Asp

115

<210> 23

<211> 523

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesized peptide

<400> 23

Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro

1 5 10 15

Arg Gly Ser His Met Ala Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys

20 25 30

Ala Lys Ala Cys Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg

35 40 45

Met Ser Val Asp Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu

50 55 60

His Tyr Ser Met Val Leu Glu Gly Gly Asn Asp Ala Leu Lys Leu Ala

65 70 75 80

Ile Asp Asn Ala Leu Ser Ile Thr Ser Asp Gly Leu Thr Ile Arg Leu

85 90 95

Glu Gly Gly Val Glu Pro Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg

100 105 110

Gln Ala Arg Gly Ser Trp Ser Leu Asn Trp Leu Val Pro Ile Gly His

115 120 125

Glu Lys Pro Ser Asn Ile Lys Val Phe Ile His Glu Leu Asn Ala Gly

130 135 140

Asn Gln Leu Ser His Met Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp

145 150 155 160

Glu Leu Leu Ala Lys Leu Ala Arg Asp Ala Thr Phe Phe Val Arg Ala

165 170 175

His Glu Ser Asn Glu Met Gln Pro Thr Leu Ala Ile Ser His Ala Gly

180 185 190

Val Ser Val Val Met Ala Gln Thr Gln Pro Arg Arg Glu Lys Arg Trp

195 200 205

Ser Glu Trp Ala Ser Gly Lys Val Leu Cys Leu Leu Asp Pro Leu Asp

210 215 220

Gly Val Tyr Asn Tyr Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp Thr

225 230 235 240

Trp Glu Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys His

245 250 255

Asp Leu Asp Ile Lys Pro Thr Val Ile Ser His Arg Leu His Phe Pro

260 265 270

Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His Leu

275 280 285

Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln

290 295 300

Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu

305 310 315 320

Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala

325 330 335

Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu

340 345 350

Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser

355 360 365

Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala

370 375 380

Asn Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly Glu Cys

385 390 395 400

Ala Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro

405 410 415

Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser Thr

420 425 430

Arg Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala Glu Phe

435 440 445

Leu Leu Leu Leu Arg Asp Glu Leu Lys Asn Tyr Gly Glu Ala Val Ser

450 455 460

Leu Ile Leu Leu Leu Asn Thr Ser Ala Cys Met Tyr Ser Lys Asp Glu

465 470 475 480

Leu Leu Leu Val Ala Val Gly Lys Met Gln Gln Phe Arg Asp Glu Leu

485 490 495

Lys Leu Leu Leu Tyr Leu Thr Glu Leu Thr Thr Val Phe Arg Asp Glu

500 505 510

Leu Lys Leu Leu Glu His His His His His His

515 520

<210> 24

<211> 696

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthesized DNA

<400> 24

gaattcggcg tgagcgcggc ccaggaaaag atcagtttcg gcctgttagg tgtgccaacg 60

gcccaagaga ctacaagtat tcgcgaggtt ttggaagtca gtactgcaca agaaaacagt 120

ccatttatgt taggcgcgag tgccacggag gaaaaaacgt ctttgcgcct gggggcaagc 180

acaacgcagg agacgagttt tggcaagtgt ttacgtccac atggggtttc tgcagcccaa 240

gggacgactc catttcgcgg tgtcagtaca acgcaagaaa acacgagttt tggtcgtgtc 300

ccaacggcac aagagaacgt gtcttttggc ctgcatggtg ttccagcagc gcaaaagacg 360

aacagcttcg gtggcgttcc aacggcacaa gaaaacatta gttttaagga ggttagtgcc 420

acgcaacgtg aaatcccatt ccgttgttta cgcccacacg gggttagcac agcccaggag 480

actccatttc gcggggtgag tactgcccag gagacgatcc cattccgtgg ggtttctgca 540

acgcatgaaa acatcagttt tgggtgtttg cgtccacatg gtgtcagcgc cgcacaggaa 600

tctattccaa tccgtctggg cgcgagcgca gcccaagaga ataccagttt tcgcgggaca 660

ccagcggcac aggagaaaat cccattggaa ctcgag 718

<210> 25

<211> 230

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesized peptide

<400> 25

Gly Pro Gly Val Ser Ala Ala Gly Gly Leu Ile Ser Pro Gly Leu Leu

1 5 10 15

Gly Val Pro Thr Ala Gly Gly Thr Thr Ser Ile Ala Gly Val Leu Gly

20 25 30

Val Ser Thr Ala Gly Gly Ala Ser Pro Pro Met Leu Gly Ala Ser Ala

35 40 45

Thr Gly Gly Leu Thr Ser Leu Ala Leu Gly Ala Ser Thr Thr Gly Gly

50 55 60

Thr Ser Pro Gly Leu Cys Leu Ala Pro His Gly Val Ser Ala Ala Gly

65 70 75 80

Gly Thr Thr Pro Pro Ala Gly Val Ser Thr Thr Gly Gly Ala Thr Ser

85 90 95

Pro Gly Ala Val Pro Thr Ala Gly Gly Ala Val Ser Pro Gly Leu His

100 105 110

Gly Val Pro Ala Ala Gly Leu Thr Ala Ser Pro Gly Gly Val Pro Thr

115 120 125

Ala Gly Gly Ala Ile Ser Pro Leu Gly Val Ser Ala Thr Gly Ala Gly

130 135 140

Ile Pro Pro Ala Cys Leu Ala Pro His Gly Val Ser Thr Ala Gly Gly

145 150 155 160

Thr Pro Pro Ala Gly Val Ser Thr Ala Gly Gly Thr Ile Pro Pro Ala

165 170 175

Gly Val Ser Ala Thr His Gly Ala Ile Ser Pro Gly Cys Leu Ala Pro

180 185 190

His Gly Val Ser Ala Ala Gly Gly Ser Ile Pro Ile Ala Leu Gly Ala

195 200 205

Ser Ala Ala Gly Gly Ala Thr Ser Pro Ala Gly Thr Pro Ala Ala Gly

210 215 220

Gly Leu Ile Pro Leu Gly

225 230

<210> 26

<211> 306

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthesized DNA

<400> 26

gaattccttc tcgagttgct ccgcgatgaa ctaaaagtgg agtattcttt tattttcctg 60

gacgagtacc ttctcctctc cactgtgacg gtgatcttcc tgaaggatga gcttttattg 120

ctcccggagt cgcgcaaaaa gctggagaag gcgcttatcg cctgggcacg tgacgaattg 180

aagctcctcc tcctactgaa agacgagctt tacgagccgc gcgactcgta cttcctgctc 240

ctgctgctcc gcgatgaact gaaacgcgtc gataacgcgc tgctgaagtt cctcctgctc 300

gtcgac 316

<210> 27

<211> 102

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesized peptide

<400> 27

Glu Phe Leu Leu Glu Leu Leu Arg Asp Glu Leu Lys Val Glu Tyr Ser

1 5 10 15

Phe Ile Phe Leu Asp Glu Tyr Leu Leu Leu Ser Thr Val Thr Gly Ile

20 25 30

Phe Leu Lys Asp Glu Leu Leu Leu Leu Pro Glu Ser Arg Lys Lys Leu

35 40 45

Glu Lys Ala Leu Ile Ala Trp Ala Arg Asp Glu Leu Lys Leu Leu Leu

50 55 60

Leu Leu Lys Asp Glu Leu Tyr Glu Pro Arg Asp Ser Tyr Phe Leu Leu

65 70 75 80

Leu Leu Leu Arg Asp Glu Leu Lys Arg Val Asp Asn Ala Leu Leu Lys

85 90 95

Phe Leu Leu Leu Val Asp

100

<210> 28

<211> 682

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesized peptide

<400> 28

Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro

1 5 10 15

Arg Gly Ser His Met Ala Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys

20 25 30

Ala Lys Ala Cys Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg

35 40 45

Met Ser Val Asp Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu

50 55 60

His Tyr Ser Met Val Leu Glu Gly Gly Asn Asp Ala Leu Lys Leu Ala

65 70 75 80

Ile Asp Asn Ala Leu Ser Ile Thr Ser Asp Gly Leu Thr Ile Arg Leu

85 90 95

Glu Gly Gly Val Glu Pro Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg

100 105 110

Gln Ala Arg Gly Ser Trp Ser Leu Asn Trp Leu Val Pro Ile Gly His

115 120 125

Glu Lys Pro Ser Asn Ile Lys Val Phe Ile His Glu Leu Asn Ala Gly

130 135 140

Asn Gln Leu Ser His Met Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp

145 150 155 160

Glu Leu Leu Ala Lys Leu Ala Arg Asp Ala Thr Phe Phe Val Arg Ala

165 170 175

His Glu Ser Asn Glu Met Gln Pro Thr Leu Ala Ile Ser His Ala Gly

180 185 190

Val Ser Val Val Met Ala Gln Thr Gln Pro Arg Arg Glu Lys Arg Trp

195 200 205

Ser Glu Trp Ala Ser Gly Lys Val Leu Cys Leu Leu Asp Pro Leu Asp

210 215 220

Gly Val Tyr Asn Tyr Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp Thr

225 230 235 240

Trp Glu Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys His

245 250 255

Asp Leu Asp Ile Lys Pro Thr Val Ile Ser His Arg Leu His Phe Pro

260 265 270

Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His Leu

275 280 285

Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln

290 295 300

Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu

305 310 315 320

Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala

325 330 335

Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu

340 345 350

Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser

355 360 365

Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala

370 375 380

Asn Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly Glu Cys

385 390 395 400

Ala Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro

405 410 415

Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser Thr

420 425 430

Arg Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala Glu Phe

435 440 445

Gly Val Ser Ala Ala Gln Glu Lys Ile Ser Phe Gly Leu Leu Gly Val

450 455 460

Pro Thr Ala Gln Glu Thr Thr Ser Ile Arg Glu Val Leu Glu Val Ser

465 470 475 480

Thr Ala Gln Glu Asn Ser Pro Phe Met Leu Gly Ala Ser Ala Thr Glu

485 490 495

Glu Lys Thr Ser Leu Arg Leu Gly Ala Ser Thr Thr Gln Glu Thr Ser

500 505 510

Phe Gly Lys Cys Leu Arg Pro His Gly Val Ser Ala Ala Gln Gly Thr

515 520 525

Thr Pro Phe Arg Gly Val Ser Thr Thr Gln Glu Asn Thr Ser Phe Gly

530 535 540

Arg Val Pro Thr Ala Gln Glu Asn Val Ser Phe Gly Leu His Gly Val

545 550 555 560

Pro Ala Ala Gln Lys Thr Asn Ser Phe Gly Gly Val Pro Thr Ala Gln

565 570 575

Glu Asn Ile Ser Phe Lys Glu Val Ser Ala Thr Gln Arg Glu Ile Pro

580 585 590

Phe Arg Cys Leu Arg Pro His Gly Val Ser Thr Ala Gln Glu Thr Pro

595 600 605

Phe Arg Gly Val Ser Thr Ala Gln Glu Thr Ile Pro Phe Arg Gly Val

610 615 620

Ser Ala Thr His Glu Asn Ile Ser Phe Gly Cys Leu Arg Pro His Gly

625 630 635 640

Val Ser Ala Ala Gln Glu Ser Ile Pro Ile Arg Leu Gly Ala Ser Ala

645 650 655

Ala Gln Glu Asn Thr Ser Phe Arg Gly Thr Pro Ala Ala Gln Glu Lys

660 665 670

Ile Pro Leu Glu His His His His His His

675 680

<210> 29

<211> 554

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesized peptide

<400> 29

Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro

1 5 10 15

Arg Gly Ser His Met Ala Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys

20 25 30

Ala Lys Ala Cys Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg

35 40 45

Met Ser Val Asp Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu

50 55 60

His Tyr Ser Met Val Leu Glu Gly Gly Asn Asp Ala Leu Lys Leu Ala

65 70 75 80

Ile Asp Asn Ala Leu Ser Ile Thr Ser Asp Gly Leu Thr Ile Arg Leu

85 90 95

Glu Gly Gly Val Glu Pro Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg

100 105 110

Gln Ala Arg Gly Ser Trp Ser Leu Asn Trp Leu Val Pro Ile Gly His

115 120 125

Glu Lys Pro Ser Asn Ile Lys Val Phe Ile His Glu Leu Asn Ala Gly

130 135 140

Asn Gln Leu Ser His Met Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp

145 150 155 160

Glu Leu Leu Ala Lys Leu Ala Arg Asp Ala Thr Phe Phe Val Arg Ala

165 170 175

His Glu Ser Asn Glu Met Gln Pro Thr Leu Ala Ile Ser His Ala Gly

180 185 190

Val Ser Val Val Met Ala Gln Thr Gln Pro Arg Arg Glu Lys Arg Trp

195 200 205

Ser Glu Trp Ala Ser Gly Lys Val Leu Cys Leu Leu Asp Pro Leu Asp

210 215 220

Gly Val Tyr Asn Tyr Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp Thr

225 230 235 240

Trp Glu Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys His

245 250 255

Asp Leu Asp Ile Lys Pro Thr Val Ile Ser His Arg Leu His Phe Pro

260 265 270

Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His Leu

275 280 285

Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln

290 295 300

Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu

305 310 315 320

Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala

325 330 335

Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu

340 345 350

Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser

355 360 365

Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala

370 375 380

Asn Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly Glu Cys

385 390 395 400

Ala Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro

405 410 415

Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser Thr

420 425 430

Arg Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala Glu Phe

435 440 445

Leu Leu Glu Leu Leu Arg Asp Glu Leu Lys Val Glu Tyr Ser Phe Ile

450 455 460

Phe Leu Asp Glu Tyr Leu Leu Leu Ser Thr Val Thr Gly Ile Phe Leu

465 470 475 480

Lys Asp Glu Leu Leu Leu Leu Pro Glu Ser Arg Lys Lys Leu Glu Lys

485 490 495

Ala Leu Ile Ala Trp Ala Arg Asp Glu Leu Lys Leu Leu Leu Leu Leu

500 505 510

Lys Asp Glu Leu Tyr Glu Pro Arg Asp Ser Tyr Phe Leu Leu Leu Leu

515 520 525

Leu Arg Asp Glu Leu Lys Arg Val Asp Asn Ala Leu Leu Lys Phe Leu

530 535 540

Leu Leu Val Glu His His His His His His

545 550

Claims (9)

1. A feed additive comprising:

a bacterial extract comprising a plurality of bacterial inclusions, wherein the bacterial inclusions comprise a polypeptide comprising:

a translocation peptide having the sequence DLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHY for translocation; and

a binding epitope, wherein the binding epitope is a chicken newcastle disease virus epitope having the amino acid sequence of SEQ ID NO: 19.

2. The feed additive of claim 1, wherein the translocation peptide is located at the N-terminus of the polypeptide and the binding epitope is located at the C-terminus of the polypeptide.

3. The feed additive according to claim 1, wherein the polypeptide further comprises: a linker polypeptide located between the translocation peptide and the binding epitope.

4. A feed additive according to claim 3 wherein the linked polypeptide is an M cell target polypeptide or an intestinal epithelial target polypeptide.

5. A feed additive according to claim 3 wherein the linked polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1 to 4.

6. The feed additive according to claim 1, wherein the strain is an escherichia coli strain, a lactobacillus strain or an enterococcus strain.

7. The feed additive according to claim 1, further comprising:

0.1 to 1 weight percent of the bacterial extract;

67 to 87 weight percent talc; and

10.5 to 18 weight percent ethylcellulose.

8. The feed additive according to claim 1, further comprising: 1.5 to 15 weight percent of resistant starch.

9. A feed, comprising:

0.03 to 5 weight percent of the feed additive according to any one of claims 1 to 8; and

95 to 99.97 weight percent of feed matrix.

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