CN119265145A - Recombinant Marek's disease virus for co-expressing newcastle disease virus F gene and infectious bursal disease virus super-strong strain VP2 gene and application thereof - Google Patents
Recombinant Marek's disease virus for co-expressing newcastle disease virus F gene and infectious bursal disease virus super-strong strain VP2 gene and application thereof Download PDFInfo
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Abstract
The invention provides a recombinant Marek's disease virus for co-expressing a newcastle disease virus F gene and an infectious bursal disease virus super-strong strain VP2 gene and application thereof, belonging to the field of genetic engineering vaccines. The recombinant cloning technology is utilized to insert an expression framework containing a newcastle disease virus gene VII type strain F gene and an infectious bursal disease virus super-strong strain VP2 gene into a spacer region between Marek's disease virus genome UL45 and UL46 genes, recombinant cosmid which is obtained by inserting the newcastle disease virus F gene and the infectious bursal disease virus super-strong strain VP2 gene expression framework between Marek's disease virus genome UL45 and UL46 genes is constructed, and recombinant virus C20101 which jointly expresses the newcastle disease virus F gene and the infectious bursal disease virus super-strong strain VP2 gene is obtained by rescue. The vaccine prepared by the recombinant Marek's disease virus strain can simultaneously prevent newcastle disease, infectious bursal disease and Marek's disease, and has good market application prospect.
Description
Technical Field
The invention belongs to the field of genetic engineering vaccines, and particularly relates to a recombinant Marek's disease virus for coexpression of a newcastle disease virus F gene and an infectious bursal disease virus super-virulent strain VP2 gene and application thereof.
Background
Newcastle Disease (ND) is an acute, highly contagious, and virulent infectious disease of chickens caused by Newcastle Disease Virus (NDV). The disease is one of the most serious diseases which endanger the chicken industry at present, and is also the poultry disease which is preferentially controlled and mainly prevented in the control planning of the medium-long-term animal epidemic disease in China. The F protein coded by the NDV is glycoprotein on the NDV envelope, is a main virulence gene and host protective antigen of the NDV, and can induce the generation of neutralizing antibodies. Since the 80 s, the large-scale chicken farms in China adopted a procedure for dense immunization of ND with high frequency. However, the traditional ND vaccine is easy to be interfered by maternal antibodies and has a short immunization duration, so that the local ND vaccine can achieve an immunization effect only by multiple times of immunization, and thus the immunization pressure and the breeding burden of chickens are increased. There is an urgent need to develop novel ND vaccines that are immune-free from maternal antibodies, have long immune duration, and have good immune effects against the epidemic genotype VII NDV.
Infectious Bursal Disease (IBD) is an acute, highly contagious disease that is caused by Infectious Bursal Disease Virus (IBDV) that primarily jeopardizes chickens. The virus is transferred into China in 1979, and after 80 s, super-strong strains (vvIBDV) appear in Europe and China successively, the mortality rate of the strains is up to more than 60%, the strains cause more serious immunosuppression, and huge losses are caused for the world poultry industry. IBDV encodes VP2 protein, which is the only capsid protein of IBDV, and is also the main virulence gene and host protective antigen of IBDV, and is capable of inducing the production of neutralizing antibodies. The current vaccines used to prevent IBD are mainly conventional inactivated vaccines and attenuated live vaccines. The inactivated vaccine has short immunization duration and needs to strengthen the immunity for many times, the immune effect of the attenuated live vaccine is easy to be interfered by parent antibody, and the medium-virulent live vaccine has great side effect and can cause the damage and immunosuppression of the bursa of Fabricius of inoculated chickens. There is a need to develop new IBD vaccines for the control of ultra-virulent strains of IBDV that are safe, free from maternal antibodies and have a long immunization duration.
Marek's Disease Virus (MDV) can cause a highly infectious, lymphoproliferative and immunosuppressive tumor disease of chicken, wherein a serum 1 type MDV attenuated live vaccine CVI988 strain is widely used for preventing and controlling Marek's disease of chicken at home and abroad. The MDV genome is large, and the replication nonessential genes for inserting or replacing exogenous genes are large, so that the MDV genome is an ideal viral vector for constructing recombinant live vector vaccines. The recombinant virus vaccine based on the MDV vector can effectively overcome the defect that the immune effect of the inactivated vaccine and the attenuated live vaccine is easily interfered by the maternal antibody. Meanwhile, MDV is toxic for life after inoculation, and can continuously express exogenous protein antigen and induce antibody production, so that the organism can generate lasting immunity.
Disclosure of Invention
The invention aims to provide a recombinant Marek's disease virus which co-expresses a newcastle disease virus F gene and an infectious bursal disease virus super-virulent strain VP2 gene.
The invention also aims to provide a construction method of the recombinant Marek's disease virus for coexpression of the newcastle disease virus F gene and the infectious bursal disease virus super-virulent strain VP2 gene.
The invention also aims to provide an application of the recombinant Marek's disease virus for coexpression of the newcastle disease virus F gene and the infectious bursal disease virus super-virulent strain VP2 gene.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
A recombinant Marek's disease virus for co-expressing the F gene of newcastle disease virus and the VP2 gene of the super-strong infectious bursal disease virus is obtained by inserting an expression frame CAGW-F2AV2 containing chicken beta actin promoter, the F gene of newcastle disease virus and the VP2 gene coding sequence of the super-strong infectious bursal disease virus, a post-transcriptional regulatory sequence of woodchuck hepatitis virus and a rabbit beta globulin polyadenylation sequence into Marek's disease virus genome.
The coding sequence F2AV2 is obtained by connecting a coding sequence of a newcastle disease virus F gene and a coding sequence of an infectious bursal disease virus super-strong strain VP2 gene by a coding sequence of a porcine teschovirus 2A self-cleaving peptide, and the nucleotide sequence of the coding sequence F2AV2 is shown as SEQ ID NO. 1.
The expression frame CAGW-F2AV2 is obtained by cloning F2AV2 between chicken beta actin promoter and rabbit beta globulin polyadenylation sequence of pCAGGS vector, inserting the regulatory sequence after the transcription of woodchuck hepatitis virus between F2AV2 sequence and rabbit beta globulin polyadenylation sequence, the nucleotide sequence of expression frame CAGW-F2AV2 is shown as SEQ ID NO. 2.
The recombinant Marek's disease virus is obtained by co-transfecting CEF cells with recombinant cosmid C1, C2, C3, C5, C6 containing Marek's disease virus CVI988 strain genomic DNA fragment and recombinant cosmid C4-45-F2AV2 containing newcastle disease virus F gene and infectious bursal disease virus super-strong strain VP2 gene expression frame, and is obtained by virus rescue, wherein the recombinant cosmid C1, C2, C3, C5, C6 respectively contains Marek's disease virus CVI988 strain genome nucleotide fragments of 1-37644, 29969-68579, 60962-99647, 115670-152340 and 143712-178311, and the recombinant cosmid C4-45-F2AV2 is obtained by inserting an expression frame CAGW-F2 between CVI988 strain genome UL45 and UL46 gene on the basis of C4, and the recombinant cosmid C4 contains CVI988 strain nucleotide fragment of 415-182.
The recombinant Marek's disease virus which jointly expresses the F gene of the newcastle disease virus and the VP2 gene of the ultra-strong infectious bursal disease virus is applied to the preparation of vaccines of newcastle disease, infectious bursal disease and Marek's disease.
The invention utilizes recombinant cloning technology to insert an expression frame containing a newcastle disease virus gene VII type strain F gene and an infectious bursal disease virus super-strong strain VP2 gene into a spacer region between a Marek's disease virus CVI988 strain genome UL45 and UL46 gene, so as to construct a recombinant Marek's disease virus vaccine strain C20101 which jointly expresses the newcastle disease virus F gene and the infectious bursal disease virus super-strong strain VP2 gene. After the recombinant virus vaccine strain is immunized on specific pathogen-free (SPF) chickens, the SPF chickens can obtain immune protection on newcastle disease viruses, infectious bursal disease viruses and Marek's disease viruses, and can be used for preparing newcastle disease, infectious bursal disease and Marek's disease vaccines.
The vaccine prepared by the recombinant Marek's disease virus strain can simultaneously provide multiple protection against infection of newcastle disease virus, infectious bursal disease virus and Marek's disease virus, and has good market application prospect.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention. In the drawings:
FIG. 1 is a diagram showing the PCR identification results of recombinant cosmid C4-45-F2AV2 expressing NDV F gene and vvIBDV VP2 gene, wherein M is DL5000 Marker,1 is the amplification result of recombinant cosmid C4-45-F2AV2, and 2 is the amplification result of parent cosmid C4;
FIG. 2 is a graph of plaque lesions generated on CEF for recombinant virus C20101;
FIG. 3 is a graph of PCR identification results of genomic DNA of recombinant virus C20101, wherein M is DL5000 Marker,1 is parental virus CVI988,2 is ddH 2 O for recombinant virus 20101,3, and 4 is recombinant cosmid C4-45-F2AV2;
FIG. 4 is a diagram showing the detection of F and VP2 protein expression in CEF infected with recombinant virus C20101 by indirect immunofluorescence assay;
FIG. 5 is a graph of replication kinetics of recombinant virus C20101 on CEF cells;
FIG. 6 is a diagram of PCR test results for genetic stability of recombinant virus C20101, wherein M is DL5000 Marker,1 is ddH 2 O,2 is CVI988,3 is C20101-F5,4 is C20101-F10,5 is C20101-F15, and 6 is C20101-F20;
FIG. 7 is a graph of the results of indirect immunofluorescence assay of recombinant virus C20101 of generation 20 expressing F and VP2 proteins;
FIG. 8 is a graph of survival after immunization of chicken with recombinant virus C20101 against virulent NDV;
FIG. 9 is a graph of survival after immunization of a chicken with recombinant virus C20101 against virulent IBDV supervirulent.
Detailed Description
The following detailed description is exemplary and is intended to provide further explanation of the invention. It will be understood by those skilled in the art that various changes and substitutions can be made in the details and form of the technical solution of the present invention without departing from the spirit and scope of the invention, but these changes and substitutions fall within the scope of the present invention.
The test methods used in the following examples are conventional methods unless otherwise specified.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
EXAMPLE 1 construction and identification of recombinant MDV expressing NDV F and vvIBDV VP2 genes
1.1 Establishment of serum 1 type MDV attenuated live vaccine CVI988 strain multi-fragment cosmid rescue system
Extracting genome DNA of a CVI988 strain of the serum 1-type MDV attenuated live vaccine, and cloning the genome DNA of the CVI988 strain into a pCC1Fos vector in a segmented manner according to the CopyControlFosmid Library Production Kit kit. According to the sequencing result of the recombinant cosmid tail end, 6 recombinant cosmids C1, C2, C3, C4, C5 and C6 cloned with CVI988 strain genome DNA fragments and capable of being spliced to cover the complete CVI988 genome are selected. Wherein, C1 comprises a nucleotide fragment of CVI988 genome 1-37644, C2 comprises a nucleotide fragment of CVI988 genome 29969-68579, C3 comprises a nucleotide fragment of CVI988 genome 60962-99647, C4 comprises a nucleotide fragment of CVI988 genome 91415-126182, C5 comprises a nucleotide fragment of CVI988 genome 115670-152340, and C6 comprises a nucleotide fragment of CVI988 genome 143712-178311. The genome sequence of the MDV attenuated live vaccine CVI988 strain has a GenBank accession number of DQ530348. The 6 recombinant cosmids cloned with CVI988 genomic DNA fragment were extracted and co-transfected into Chick Embryo Fibroblasts (CEFs) by the calcium phosphate transfection method. Cytopathic effect was observed 4-5 days after transfection, i.e., parental strain CVI988 was obtained by rescue.
1.2 Construction and identification of recombinant cosmids expressing NDV F and vvIBDV VP2 genes
According to the coding region sequence of the strain GM strain (GenBank accession number DQ 486859) F gene of the NDV gene VII, synthesizing a target gene NDF by optimizing and designing a chicken codon, and adding a self-cleaving peptide sequence of porcine teschovirus 2A (P2A) at the C end of the target gene NDF. The target gene IBDV2 is synthesized by optimizing and designing chicken codons according to the gene sequence of the IBDV supervirulent strain UPM766 (GenBank accession number MT 505341) VP 2. The F2AV2 (SEQ ID NO. 1) gene of interest comprising the F gene and VP2 gene coding sequences is obtained by fusion PCR amplification using primers NDFP1F, NDV2P1R, NDV P2F and V2P2R, wherein the F gene and VP2 gene coding sequences are linked by a self-cleaving peptide P2A coding sequence. Cloning the purified PCR product into the downstream of chicken beta actin promoter of pCAGGS carrier via EcoRI and ClaI cleavage sites, and inserting the transcriptional regulatory sequence of woodchuck hepatitis virus between the target gene F2AV2 and rabbit beta globulin polyadenylation sequence to obtain recombinant plasmid pCAGW-F2AV2 for co-expressing NDV F gene and IBDV VP2 gene. Sequencing the recombinant plasmid by using primers CAGF and CAGR, and finding that the inserted target gene sequence is correct.
According to Counter Selection BAC Modification Kit kit, F2AV2 gene expression frame CAGW-F2AV2 (SEQ ID NO. 2) was cloned into the spacer region between the MDV genome UL45 and UL46 genes in recombinant cosmid C4 by Red/ET recombination method, and recombinant cosmid C4-45-F2AV2 was constructed to obtain coexpression of NDV F gene and vvIBDV VP2 gene. And (3) using a target gene primer vvIBDVP and a target gene downstream homology arm primer MDV46R to identify whether recombinant cosmid C4-45-F2AV2 is inserted into a target gene expression frame or not through PCR, and sequencing and identifying a PCR product. The results showed that the obtained PCR product had a length of 2760 bp (FIG. 1) and a size consistent with the expectations. Sequencing results show that the PCR product contains VP2 gene sequence and downstream homology arm sequence of target gene expression cassette, and the sequence is correct. The negative control sample (parental cosmid C4) had no insert of the gene of interest and the PCR result was negative. The above results indicate that recombinant cosmid C4-45-F2AV2 was constructed correctly.
TABLE 1 PCR primers for construction and identification of recombinant cosmids of F gene and VP2 gene
1.3 Rescue and identification of recombinant MDV expressing NDV F and vvIBDV VP2 genes
Recombinant cosmid C4-45-F2AV2 and other parental cosmids cloned with the genomic fragment of MDV CVI988 strain were extracted using a plasmid extraction kit. The recombinant cosmid C4-45-F2AV2 and the parental cosmid C1, C2, C3, C5 and C6 are co-transfected into CEF cells by a calcium phosphate transfection method, viruses are harvested after 4-5 days of culture and plaque lesions appear, and are continuously passaged and preserved in the CEF cells, so that the recombinant viruses with the F2AV2 expression frame inserted between the UL45 and UL46 genes of the MDV genome are obtained through rescue, and are named as recombinant viruses C20101. Among them, the plaque lesions generated on CEF by the 5 th generation recombinant virus C20101 are shown in FIG. 2.
The 5 th generation recombinant virus genome DNA is extracted, and the parental virus CVI988 genome DNA is set as a control. The recombinant virus was identified by PCR using the target gene specific primer vvIBDVP F and the target gene downstream homology arm primer MDV46R, and the PCR product was further analyzed by sequencing. The PCR results showed that the length of the PCR product obtained by amplification of the recombinant virus was 2760 bp, which was consistent with the expected size (FIG. 3). Sequencing results show that the PCR product contains the vvIBDV VP2 gene sequence and the downstream homology arm sequence of the target gene expression cassette, and the sequences are correct. The parental virus CVI988 has no target gene insertion, and the PCR result is negative. The result shows that the target gene F2AV2 is correctly inserted into the genome of the strain CVI988 of the serum 1 MDV attenuated live vaccine, and the recombinant virus C20101 is correctly constructed.
EXAMPLE 2 in vitro biological Property analysis of recombinant MDV expressing NDV F and vvIBDV VP2 genes
2.1 Detection of recombinant virus C20101 expression F protein and VP2 protein
Recombinant virus C20101 and parent virus CVI988 are respectively inoculated with CEF cells, after plaque lesions appear in the cells after 3-4 days of culture, the cells are fixed by absolute ethyl alcohol, NDV positive serum and VP2 protein monoclonal antibodies are respectively used as primary antibodies, FITC-marked rabbit anti-chicken IgG and FITC-marked goat anti-mouse IgG are used as secondary antibodies, an indirect immunofluorescence test is adopted to detect the expression of F protein and VP2 protein, and CEF of which the parent virus is inoculated with the virus is used as negative control. The results showed that recombinant virus C20101-infected cells were able to react with NDV positive serum and VP2 protein monoclonal antibodies, showing green fluorescent signals (fig. 4). The MDV parental virus CVI988 strain received the virus cells and the control cells without the virus received no fluorescence. The above results indicate that recombinant virus C20101 can co-express the F protein and VP2 protein in infected cells.
2.2 In vitro replication characterization of recombinant virus C20101
Recombinant virus C20101 and parental virus CVI988 were inoculated at a dose of 100 Plaque Forming Units (PFU) onto CEF in 6-well plates, and the toxic cells were collected every 24 hours after infection, for up to 144 hours after infection. The virus collected at each time point was inoculated with CEF, the number of plaques of the virus solution at each time point was measured, an in vitro replication kinetics curve was drawn, and the in vitro replication characteristics of the recombinant virus C20101 and the parent virus in CEF were analyzed. The results showed that the replication titers of both recombinant virus C20101 and parent virus CVI988 reached the highest peak at 120 hours post-infection, 9.13×10 4 PFU/ml and 9.89×10 4 PFU/ml, respectively, and there was no significant difference (P > 0.05) in the titers of recombinant virus from the parent virus at each time point after infection with CEF (fig. 5). The above results indicate that the replication characteristics of recombinant virus C20101 in CEF are consistent with the parental virus CVI988 strain.
2.3 Genetic stability detection of recombinant virus C20101
The 5 th generation recombinant virus C20101 was serially passaged 20 generations on CEF. And selecting 5 th, 10 th, 15 th and 20 th generation cell toxins, extracting viral genome DNA, carrying out PCR identification and sequencing, and detecting the genetic stability of a target gene sequence in a recombinant viral genome. Meanwhile, the 20 th generation recombinant virus is inoculated to CEF cells, and an indirect immunofluorescence test is carried out by using NDV positive serum and VP2 monoclonal antibody to detect the expression stability of the target genes F and VP 2. The PCR identification result shows that the target genes F and VP2 exist stably in the MDV genome, and the 5 th, 10 th, 15 th and 20 th generation recombinant viruses can be amplified to obtain target gene bands with the sizes of 2760 bp (figure 6), which accords with the expectations. And amplifying the 20 th generation recombinant virus genome DNA by using primers CAGF and CAGR, and sequencing PCR products to find that the inserted target gene sequence is correct. And (3) taking the 20 th generation recombinant virus to carry out an indirect immunofluorescence test, and detecting the expression condition of the target genes F and VP2 in the passage process. The results showed that the genes of interest F and VP2 were stably expressed after continuous transfer of recombinant virus C20101 on CEF cells to passage 20 (FIG. 7). The result shows that the recombinant virus C20101 has good genetic stability.
Example 3 safety and immunogenicity assays for recombinant Virus C20101
3.1 Safety test of recombinant virus C20101
The 5th generation recombinant virus C20101 was inoculated at 2000 PFU/dose to 1 day old SPF chickens and clinical symptoms of each group were observed daily after inoculation. And (3) 28 days after inoculation, randomly selecting 5 test chickens in each group, weighing, evaluating the influence of recombinant virus C20101 on the growth and development of the test chickens, cutting off the 5 selected chickens in each group, collecting organs such as bursa of Fabricius, thymus, spleen, liver and the like, weighing, and observing whether atrophy or swelling symptoms exist or not. As a result, the recombinant virus C20101 does not cause adverse clinical reaction of the test chicken after being inoculated with the SPF chicken, and the test chicken feeds and drinks normally. The results of weighing and sectioning after 28 days of inoculation show that the weight of the recombinant virus C20101 inoculated group test chicken has no obvious difference from that of a normal non-inoculated control group, and the test chicken is sectioned and inspected, and organs such as bursa of Fabricius, thymus, spleen, liver and the like are collected, so that the organs are normal and no obvious clinical lesions are found. The above results indicate that recombinant virus C20101 is non-pathogenic to SPF chickens.
3.2 Immunoprotection test of recombinant virus C20101 against NDV and IBDV supervirulence
60 SPF chicks with the age of 1 day are randomly divided into 3 groups, 20 chicks are selected from each group, the recombinant virus C20101 is inoculated subcutaneously on the back of the neck at the dose of 2000 PFU per dose, the parental virus CVI988 is inoculated on the same dose of group 2, and the non-immunized group 3 serves as a blank control group. And taking 10 chickens with strong virus NDV in group 1 and group 2 respectively 28 days after immunization, observing for 14 days after virus challenge, recording clinical symptoms of the test chickens, and counting survival conditions of the chickens in each group. The remaining 10 chickens in group 1 and group 2 were challenged with ultra-virulent IBDV respectively, observed for 7 days after challenge, recorded for the clinical symptoms of the test chickens, counted for survival, and examined for the pathological changes of bursa of Fabricius by dissecting the test chickens for 7 days after challenge. Group 3 was not challenged as a healthy control group.
The results showed that all healthy survived during the observation period after 10 chickens vaccinated with recombinant virus C20101 had been virulent NDV and all died after 10 chickens vaccinated with parental virus CVI988 had been virulent NDV (fig. 8). All healthy survived during the observation period after 10 chickens vaccinated with recombinant virus C20101 had virus-challenged vvIBDV, the bursa of Fabricius had no obvious lesions, and 9 chickens died after 10 chickens vaccinated with parental virus CVI988 had virus-challenged vvIBDV (FIG. 9), with atrophy, bar bleeding or yellow jelly-like infiltration lesions all occurring in the bursa of Fabricius. The result shows that the recombinant virus C20101 has good immune protection effect on the NDV virulent and the IBDV virulent.
3.3 Immunoprotection test of recombinant virus C20101 against MDV virulence
40 SPF chicks of 1 day old were randomly divided into two groups of 20 chicks. Group 1 was subcutaneously vaccinated with recombinant virus C20101 at 2000 PFU/dose cervical back, and group 2 was not vaccinated as a control group. And 7 days after inoculation, the test chickens are respectively subjected to intraperitoneal injection of MDV strong toxin, clinical symptoms of the test chickens are observed after toxin attack until 80 days after toxin attack, and the morbidity and mortality of the test chickens are counted. And (5) timely cutting the dead chicken, finishing the observation period, cutting all test chicken, and recording pathological changes of all organs.
The results show that 16 chickens die after the non-immune control group attacks the toxic MDV, the surviving chickens do not have typical MD clinical symptoms in the observation period, and the split examination shows that 18 chickens have MD characteristic lesions such as hepatosplenomegaly, visceral tumor and the like, and the MD positive rate is 90%. The split inspection shows that two chickens in the group have hepatomegaly lesions, the other 18 chickens do not have MD characteristic lesions, and the MD positive reduction rate of the recombinant virus C20101 immune group is 89%. The result shows that the recombinant virus C20101 has good immune protection effect on MDV virulence.
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