CN115896151B - Construction method and application of food-grade recombinant duck tembusu virus truncated E gene lactococcus lactis vector - Google Patents
Construction method and application of food-grade recombinant duck tembusu virus truncated E gene lactococcus lactis vector Download PDFInfo
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Abstract
The invention discloses a construction method and application of a food-grade recombinant duck tembusu virus truncated E gene lactococcus lactis vector; the technical scheme is that the E gene is synthesized after lactic acid bacteria codon optimization, then a primer is designed to amplify domainI-domainII region fragment of the E gene, the fragment is used as a target gene to be subjected to homologous recombination with plasmid pNZ8149 to construct recombinant plasmid, the recombinant plasmid is electrically transformed into NZ3900 competent bacteria, positive clone strains are selected and cultured, and the positive plasmid is extracted for PCR identification and sequencing analysis to obtain the recombinant vector; then using an inducer to induce the expression of target protein, and detecting recombinant protein; optimizing the optimal induction expression condition of protein expression, and expressing the obtained target protein in cells by using soluble protein and inclusion body protein; the method has the advantages of high production safety, low cost, no stress in the immune process and capability of being applied to the domestic duck cultivation in a large scale for resisting the live carrier vaccine of the duck tembusu virus.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a construction method of a food-grade recombinant duck tembusu virus truncated E gene lactococcus carrier, and also relates to application of the food-grade recombinant duck tembusu virus truncated E gene lactococcus carrier.
Background
Duck tembusu virus disease is an infectious disease that is caused by infection of ducks of different ages and other birds such as geese, chickens, sparks with Duck tembusu virus (DTMUV). Studies have shown that sparrows and mosquitoes are important vehicles for the transmission of the disease. DTMUV is a member of the flaviviridae, flaviviridae, mosquito vector virus, and Entaya virus group. The DTMUV genome is a positive strand RNA of about 11kb in length, comprising 1 large Open Reading Frame (ORF) encoding a polyprotein precursor of 3425 amino acids in length [1]. The structural components of the DTMUV virus particles have 3 structural proteins: nucleocapsid protein (C), membrane protein precursor (prM) and envelope protein (E) and 7 nonstructural proteins. The E protein is an important immunogenic protein, the structure of the E protein can be divided into domainI, domainII and domainIII regions, and researches show that the three regions have antigenic determinants capable of inducing the generation of systemic neutralizing antibodies, and the E protein is the preferred antigenic protein for researching DTMUV genetic engineering vaccines.
The greatest advantage of developing genetic engineering vaccine is high safety, and the defects of strong virulence return, high immunity injection and the like are avoided. The E protein is used for various targets for developing genetic engineering vaccines, such as DTMUV E protein liposome vaccine, VLP E-HA2 virus-like particle vaccine, rAD-E recombinant adenovirus vaccine, E-ch recombinant duck enteritis virus live vector vaccine, prM and E protein attenuated salmonella typhimurium oral DNA vaccine. The research utilizes food-grade lactococcus lactis NZ3900 and a matched plasmid pNZ8149 thereof developed by the NIZO food research in the Netherlands, which are not used in the development of duck Tembusu virus genetic engineering vaccines, the strain and the matched plasmid thereof are operation systems which are matched with the unique pair of the existing lactobacillus expression exogenous proteins and take lactose as a unique carbon source to screen positive clones, do not contain resistance genes at all, express various viral proteins and obtain good immune effects. Compared with the lactic acid bacteria planted in the intestinal tracts of animal organisms, the lactic acid lactococcus is only survived for 5-7 days in the gastrointestinal tracts, and researches prove that the recombinant lactic acid lactococcus vaccine can completely release the antigen into the organisms to induce immunity through repeated immunity within a plurality of weeks, and can not cause immune tolerance problem. In view of this, food-grade lactococcus lactis NZ3900 and its companion plasmid used in the experiments are suitable for use in delivery live vectors capable of producing proteins of interest in later studies, and do not produce any oral lactobacillus mucosal immune delivery vaccine of harmful products. Strain NZ3900 has been shown to be useful for overproducing active exogenous proteins.
Disclosure of Invention
The invention aims to provide a construction method and application of a food-grade recombinant duck tembusu virus truncated E gene lactococcus lactis vector, the expression quantity of its exogenous protein is considerable, and the expression condition of the protein is optimized, and the adopted inducer nisin concentration dose is only 1ng/mi, so that the cost is low, and the recombinant duck tembusu virus truncated E gene lactococcus lactis vector can be used as an oral vaccine of duck tembusu virus.
Therefore, the technical scheme provided by the invention is as follows:
the construction method of the food-grade recombinant duck tembusu virus truncated E gene lactococcus lactis vector sequentially comprises the following steps:
1) Optimized synthesis of full-length E gene fragments
Inserting two restriction sites of Nco I and Sac I at two ends of 1502bp of the full-length sequence of the E gene, loading the two restriction sites into pUC57 plasmid, transforming into Top10 clone strain, and naming the recombinant plasmid as pUC57-E;
2) Construction of NZ3900-pNZ8149-DI-DII recombinant expression vector
2.1 Primer design)
Designing a primer for amplifying the DI-DII fragment, wherein the nucleotide sequence gene sequence of the DI-DII is shown in SEQ ID NO: 1.
2.2 Acquisition of fragments of interest
Amplifying the target DI-DII gene by taking the pUC57-E of the step 1) as a template for PCR amplification reaction, observing the size of the fragment by electrophoresis, cutting off the target fragment with correct size and carrying out glue recovery for standby;
2.3 Extraction of pNZ8149 plasmid
Electrotransferring the plasmid pNZ8149 into lactobacillus NZ3900 competent bacteria, extracting the plasmid pNZ8149, and performing electrophoresis observation;
2.4 Preparation of NZ3900 competent bacteria
2.5 Cleavage and recovery of pNZ8149 plasmid
Double digestion is carried out on the pNZ8149 plasmid by using NcoI and SacI, digestion is carried out overnight at 37 ℃, electrophoresis of the digested pNZ8149 is observed, and gel cutting recovery is carried out for standby after the complete digestion is confirmed;
2.6 Seamless cloning of the fragment of interest and the pNZ8149 plasmid
Carrying out homologous recombination connection reaction on the target gene after glue recovery and the pNZ8149 plasmid, and carrying out electrotransformation after the reaction system is incubated at 50 ℃ for 15 min;
2.7 Recombinant plasmid electrotransfer lactococcus lactis
Placing 40ul of lactobacillus NZ3900 competent bacteria on ice for 2-3min, adding 4ul of connection product (pNZ 8149 is added into 1 ul), standing on ice for 15min, transferring into a 0.2mm electrorotating cup for electrorotating, and rapidly resuscitating with 1ml of electrorotating resuscitating liquid prepared from ice after electrorotating; ice pre-preparing for 5min, standing at 30 ℃ for 1.5h, transferring into a centrifuge tube, centrifuging, discarding a part of supernatant in an ultra-clean bench, re-suspending thalli, taking a 50ul coating plate, inversely culturing at 30 ℃ for 18h, and selecting a monoclonal after the monoclonal grows out for culture identification;
3) Identification of recombinant plasmids
3.1 Plasmid identification
Taking out suspected positive clones, picking up monoclonal bacteria, culturing at 30 ℃ in an EM culture medium overnight, extracting plasmids from bacterial liquid the next day, and performing preliminary judgment by comparing the sizes of plasmids with the sizes of pNZ 8149;
3.2 PCR identification
Carrying out PCR identification by taking positive cloned plasmid as a template; after the PCR is completed, electrophoresis is carried out for observation;
3.3 Sequencing identification
Sequencing the extracted plasmid, and comparing the extracted plasmid with a primary gene sequence through DNAstar software;
4) Expression and analysis of exogenous target protein of recombinant lactococcus lactis
4.1 Inducible expression of recombinant lactococcus lactis exogenous target protein and protein extraction;
4.2 SDS-PAGE and Western blot to detect exogenous target protein;
4.3 Orthogonal experiments are carried out on target proteins through different Nisin concentrations, induction time and OD 600 to optimize protein expression.
Furthermore, the construction method of the food-grade recombinant duck tembusu virus truncated E gene lactococcus lactis vector comprises the following steps of: F-5'-TAAGGAGGCACTCACATGTTTTCATGTTTGGGTATGCAAAAT-3'; the sequence of the downstream primer is as follows: R-5'-GTTCAAAGAAAGCTTGAGCTCATACCTTTTAACTTTAAACC-3'.
Furthermore, the construction method of the food-grade recombinant duck tembusu virus truncated E gene lactococcus lactis vector comprises the following components in parts by volume: 2 Xbuffer 25. Mu.L; dNTP 10. Mu.L; 1. Mu.L of the upstream primer; 1. Mu.L of a downstream primer; KODFx. Mu.L; ddH 2 O11 μL; 1 μl of cDNA; total volume 50 μl;
The reaction conditions are as follows: 94 ℃,2min,98 ℃,10s,56 ℃,30s,68 ℃,30s,35 cycles, and 68 ℃ for 10min.
Furthermore, the construction method of the food-grade recombinant duck tembusu virus truncated E gene lactococcus lactis vector comprises the following steps of: culturing NZ3900 strain containing pNZ8149 plasmid with EM culture medium at 30deg.C overnight, taking 5ml of bacterial liquid the next day, centrifuging 12000g for 1min, and discarding supernatant; washing 2-3 times with 10mM Tril-HCl, pH=8.5, centrifuging at 12000g for 1min, and discarding the supernatant; adding 1ml of lysozyme with pH= 8.020mg/ml, standing at 37 ℃ for 2 hours, centrifuging 12000g for 5 minutes, and discarding the supernatant; the pellet was subjected to plasmid extraction as described in the miniplasmid kit and the extracted plasmid was observed by electrophoresis.
Furthermore, the method for constructing the food-grade recombinant duck tembusu virus truncated E gene lactococcus lactis vector comprises the following components in parts by volume: pNZ8149 30. Mu.L; ncoI 1. Mu.L; sacI 1 μl;10 Xbuffer 5. Mu.L; ddH 2 μL; totaling 50 μl.
Furthermore, the construction method of the food-grade recombinant duck tembusu virus truncated E gene lactococcus lactis vector comprises the following components in parts by volume: pNZ 8149. Mu.L; 3 mu L of target gene fragment; 2XSeamlessCloningMix 10 μl; ddH 2 0 6. Mu.L; totaling 20. Mu.L.
Further, the construction method of the food-grade recombinant duck tembusu virus truncated E gene lactococcus lactis vector comprises the following steps of:
(1) Streaking lactobacillus NZ3900 on a G-M17 plate, and culturing overnight at 30 ℃;
(2) The next day, selecting a monoclonal to a G-SGM17 culture solution, and culturing overnight at 30 ℃;
(3) Thirdly, transferring the lactobacillus NZ3900 culture solution into G-SGM17 according to the proportion of 1:10, and standing and culturing at 30 ℃ overnight;
(4) Amplifying and culturing lactobacillus culture solution NZ3900 into G-SGM17 according to the ratio of 1:8, standing and culturing at 30 ℃ until OD 600 = 0.2-0.3, centrifuging at the temperature of 4 ℃ for 10min at the speed of 8000rpm, and collecting thalli;
(5) Re-suspending the thallus with 10-20mlEPB, centrifuging at 8000rpm and 4 deg.c for 10min, and collecting thallus;
(6) The cells were resuspended with 50ml EPB+EDTA, pre-incubated with ice for 15min, then centrifuged to collect the cells, resuspended with 5-10mlEPB, centrifuged to collect the cells, and finally expanded with EPB to a final cell volume of 100:1, concentrating and resuspending thalli in proportion, and placing on ice;
(7) Then, the thalli are packaged rapidly, 40ul each is stored at-80 ℃.
Furthermore, the construction method of the food-grade recombinant duck tembusu virus truncated E gene lactococcus lactis vector adopts a furgi and shs mode, and the electric shock is 4.5ms.
Furthermore, the construction method of the food-grade recombinant duck tembusu virus truncated E gene lactococcus lactis vector comprises the steps of preparing an induced expression target protein and a protein sample, selecting a monoclonal strain, standing and culturing at 30 ℃ overnight, and activating lactobacillus according to the following steps of 1:50 is transferred into fresh EM, when OD 600 = 0.4-0.5, nisin with the final concentration of 1ng/ml is added for induction, and static culture is carried out for 3 hours at 30 ℃; after the induction, 2ml of bacterial liquid is taken for centrifugal separation of bacterial body and supernatant, a proper amount of ice-cooled 10mM Tris-hcl is used for precipitation, suspended cells with PH=8.5 are centrifugated at 8000rpm/min, and the supernatant is discarded; repeating for 2 times, re-suspending with 140 mu L of 10mMTfis-hcl containing 1mg/mL lysozyme, carrying out reaction at the pH=8.5 in a 37 ℃ incubator for 1h, centrifuging, discarding the supernatant, adding 30 mu L of 20wt% SDS, uniformly mixing and lysing the thalli, adding 70 mu L of 10mM Tris-hcl, carrying out pH=8.5, adding 50ul of 5X protein loading buffer, blowing and uniformly mixing, and carrying out boiling water bath for 10min; then, the sample was cooled in an ice box, 10. Mu.L of the sample was taken as a sample, and the remainder was kept at-80 ℃.
Further, the construction method of the food-grade recombinant duck tembusu virus truncated E gene lactococcus carrier comprises the steps of extracting protein, washing induced lactobacillus twice in PBS, re-suspending the lactobacillus in PBS with the ratio of 1:10, placing the lactobacillus on ice for ultrasonic treatment, centrifuging for 10 minutes at ultrasonic power of 200V for more than 5 seconds, stopping for 6 seconds, performing ultrasonic treatment for 30 minutes, separating supernatant and precipitate, and then adding 40ul of the lactobacillus into protein loading buffer solution, heating and boiling for 10 minutes for later use.
Further, the construction method of the food-grade recombinant duck tembusu virus truncated E gene lactococcus lactis vector comprises the following steps of: preparing 12wt% of separating gel and 5wt% of concentrating gel, assembling an electrophoresis tank, placing into an electrophoresis apparatus, adjusting the voltage to 80V for 30min after sample loading, adjusting to 120V for 1h, and dyeing the gel for 2h by using coomassie brilliant blue dye solution after electrophoresis is completed. And after the dyeing reaction is finished, adding a decoloring liquid to decolor, and observing the result.
The Westernblot detection method comprises the following steps: proteins were separated by 12wt% SDS-PAGE gel electrophoresis, transferred to PVDF membrane using wet transfer method, blocked with rapid blocking solution at room temperature for 10min, washed, incubated with murine E protein polyclonal antibody diluted 1000-fold with PBS for 1h, washed, incubated with HRP-labeled goat anti-mouse IgG for 1h, washed, and then stained with ECL chromogenic reagent dropwise for visualization on gel imaging system.
Compared with the prior art, the technical scheme provided by the invention has the following technical advantages:
The invention provides a specific primer combination, a more convenient seamless cloning method and constructs a carrier NZ3900-pNZ8149-DI which can stably and excessively express recombinant DI-DII fusion protein, the oral live carrier of the lactobacillus is food-grade, the carried pNZ8149 plasmid does not carry any resistance gene, and the oral live carrier is taken as the live carrier, so that the oral live carrier does not bring any drug resistance risk caused by transfer of the resistance gene to the environment. The expressed recombinant protein can react with polyclonal antiserum of E protein, has immunoreactivity, and can produce a large amount of active protein in a short time by optimizing three-factor three-level protein expression conditions and using nisin induction dosage of 1ng/ml required by the optimal protein expression conditions, and a living carrier can carry enough antigen protein. Compared with the common vaccine, the vaccine has the advantage of being orally taken, the immune process does not generate stress on poultry, lactic acid bacteria can promote the intestinal health of the poultry, and compared with other oral DNA live carrier vaccines, the vaccine has the advantage of carrying active antigen protein, has high biological safety, and does not have the problem of pollution of diffusion resistance genes or DNA plasmid genes. Secondly, the inducer dosage used by the carrier can induce the production of antigen active protein under the condition of 1ng/ml, greatly reduces the cost, accords with the practical production application, and finally, compared with the common vaccine, the live carrier does not need an adjuvant, and the lactobacillus has the function of serving as the adjuvant.
Drawings
FIG. 1 is a cut-away view of a fragment of interest;
FIG. 2 is a pUC57-E plasmid identification map;
FIG. 3 is an amplification diagram of a fragment of interest;
FIG. 4 is a graph showing the result of electric transformation of recombinant plasmids;
FIG. 5 is an identification chart of recombinant plasmids;
FIG. 6 is a diagram showing SDS-PAGE detection of recombinant protein expression;
FIG. 7 is a diagram of the expression of a recombinant protein detected by Westernblot;
FIG. 8 is an optimization of protein expression conditions;
FIG. 9 is a diagram showing the detection of the position of protein expression;
FIG. 10 is a map of the pNZ8149-DI-DII plasmid.
Detailed Description
The following claims are presented in further detail in connection with the detailed description, but are not to be construed as limiting the invention, and any limited number of modifications which may be made by anyone within the scope of the claims are intended to be within the scope of the invention.
Example 1
1. The plasmids and strains adopted by the application are as follows:
The L.lactis NZ3900 strain and the L.lactis expression plasmid pNZ8149 were maintained by the laboratory from which the murine E protein polyclonal antiserum was prepared and maintained.
2. The main reagents of the application are as follows:
Nisin inducer was purchased from Sigma; the Nco I, sac I endoenzymes, seamless Cloning Kit (seamless cloning kit), lysozyme, miniplasmid kit and glue recovery kit were purchased from the bi yun tian biotechnology company; quick blocking solution and universal antibody dilutions were purchased from new sirame biotechnology limited, guangzhou, HRP-goat anti-mouse 1g secondary antibody from Abbkine Scientific co.
3. The main instrument of the application is as follows:
Nucleic acid electrophoresis apparatus (Beijing six biotechnology Co., ltd.); vertical protein electrophoresis tank (Beijing six biotechnology Co., ltd.); film transfer apparatus (Beijing six biotechnology Co., ltd.); electrotransport (Bio-rad); gene amplification apparatus (Hangzhou Langmuir scientific instruments Co.); ultraviolet spectrophotometers (Shanghai instruments and electric analysis instruments Co., ltd.); low temperature high speed centrifuges (Anhui middle department of well science instruments Co., ltd.); ultrasonic cytoclasis apparatus (Shanghai Lichen technology Co., ltd.); an electric thermostatic water bath (Changzhou Australia instruments Co., ltd.); constant temperature stationary incubator (Shanghai macrolaboratory equipment Co., ltd.); biosafety cabinet (blely clean plant limited, su state); an analytical balance; thin layer gel scanner (Beijing six biotechnology Co., ltd.); gel imaging system (Beijing six biotechnology Co., ltd.); ultra-pure water purifier (Chongqing Lidi laboratory instruments Co., ltd.); horizontal decolorizing tables (Haimen Kaolinbel Co., ltd.) and the like.
4. Preparation of main solvent and reagent
(1) L-Elliker liquid medium: 20g of Tryptone, 5g of Yeast extract, 4g of NaCl, 1.5g of anhydrous sodium acetate and 0.5g of ascorbic acid are dissolved in 1L of deionized water, stirred and mixed uniformly, and autoclaved at 121 ℃ for 20min. After cooling, lactose sterilized by filtration through a 0.22mm filter was added to give a final lactose concentration of 0.5wt%.
(2) L-Elliker Medium solid Medium: 15g of agar powder is added on the basis of the L-Elliker liquid culture medium, and the mixture is autoclaved for 20min at 121 ℃. After cooling, lactose which was sterilized by filtration through a 0.22mm filter was added to give a final lactose concentration of 0.5wt%, and then poured into a plate, cooled and solidified, and after labeling, stored in a refrigerator at 4 ℃.
(3) G-M17/G-SGM17:42.3g of M17 culture medium is dissolved in 1L of deionized water, stirred and mixed uniformly, and autoclaved at 120 ℃ for 20min; G-M17: m17 is cooled and then added with glucose which is filtered and sterilized by a 0.22mm filter membrane to ensure that the glucose concentration is 0.5 weight percent; G-SGM17: the G-M17 broth was added with sterilized sucrose (final concentration 0.5M), glycine (final concentration 2.5 wt%).
(4) Phosphate Buffer (PBS): 8g of NaCl, 2PO41.42g、KH2 g of NaH PO40.27g and 0.2g of KCl are fully dissolved in 800ml of deionized water, the PH value is adjusted to 7.4, the volume is fixed to 1L, and the packaging is carried out and then the high-pressure sterilization is carried out for 20min at 121 ℃.
(5) EPB/EPB+EDTA: EPB:0.5M sucrose+10wt% glycerol was dissolved in a corresponding volume of deionized water and autoclaved at 121℃for 20min. Epb+edta: EPB+0.05M EDTA, and pH was adjusted to 8.0 with a pH meter.
(6) Electrokinetic resuscitation fluid: lactose was added to M17 at a final concentration of 0.5wt%, 20mM MgCl 2、2mM CaCl2.
(7) Nisin inducer: nisin was diluted to 10mg/ml with 0.02N HCl as Nisin stock, stored at-20℃and diluted to 1ng/ul for use, and then induced at 1:1000.
Coomassie brilliant blue staining solution: it contains 45 methanol (V/V), 10 glacial acetic acid (V/V), 45ddH 2 O (V/V), 0.05 Coomassie brilliant blue R-250 (W/V), and is stored at room temperature.
(8) Transfer buffer: 2.9g glycine, 5.8g Tris-base and 0.37g SDS were dissolved in 600ml deionized water, and the solution was stirred well, 200ml methanol was added, and the volume was adjusted to 1000ml with deionized water and stored at room temperature.
(9) 10MM Tris-hcl (pH=8.5): 1.21g of Tris solid was dissolved in 800ml of deionized water, the pH was adjusted to 8.0 using a pH meter, and then the volume was fixed to 1L and stored at 4 ℃.
The application provides a construction method of a food-grade recombinant duck tembusu virus truncated E gene lactococcus lactis vector, which specifically comprises the following steps:
1) Optimized synthesis of full-length E gene fragments
According to the gene sequence of duck Tembusu virus JM strain (GenBank accession number; JN 811559) published in GenBank, carrying out codon optimization according to host bacterium MG1363 by Nanjing tripod biotechnology limited company, synthesizing the full-length sequence 1502bp of E gene, inserting two restriction enzyme sites of Nco I and Sac I at two end points, constructing pUC57 plasmid, transforming into Top10 clone strain, and naming the recombinant plasmid as pUC57-E; identification of pUC57-E recombinant plasmid monoclonal bacteria were picked up by streaking with glycerol bacteria, and electrophoresis after shaking to extract the plasmid was shown in FIG. 2 (M2: 0.2-1.2KD DNA Marker;1,2: recombinant plasmid pUC 57-E).
2) Construction of NZ3900-pNZ8149-DI-DII recombinant expression vector
2.1 Primer design)
The following diagram is designed to intercept the target fragment (see FIG. 1), and the primers for amplifying the DI-DII fragment (Table 2-1) are designed by using the method of homologous recombination:
TABLE 2-1 primer design
2.2 Acquisition of fragments of interest
The recombinant plasmid pUC57-E was used as a template for PCR amplification, and the target DI-DII gene was amplified, and the PCR amplification target gene reaction system was as shown in Table 2-2 below.
TABLE 2-2 PCR amplification reaction System
The reaction conditions are as follows: 94 ℃,2min,98 ℃,10s,56 ℃,30s,68 ℃,30s,35 cycles, and 68 ℃ for 10min. After amplification, adding a proper amount of DNA loading buffer solution into the PCR product, uniformly mixing, and then electrophoretically observing the fragment size as shown in figure 3, and then cutting gel at the E position of the target gene for gel recovery for later use. After PCR amplification reaction, electrophoresis is shown as (FIG. 3), M2:0.2-1.2KD DNA Marker;1: the size of the DI-DII fragment with the size of the electrophoresis band meeting the expected size of the target fragment is 900bp.
2.3 Extraction of pNZ8149 plasmid
Plasmid pNZ8149 was electroporated into lactobacillus NZ3900 competent bacteria, electrorated (fungi, shs mode, showing a shock time after shock, optimal shock time of 4.5 ms). Then, the NZ3900 strain containing pNZ8149 was allowed to stand overnight at 30℃in an EM medium, and the next day, 5ml of the bacterial liquid was collected, 12000g was centrifuged for 1min, and the supernatant was discarded. The supernatant was discarded after washing 2-3 times with 10mM Tril-HCl, pH=8.5, and centrifugation at 12000g for 1 min. 1ml of 20mg/ml lysozyme (ready-to-use preparation, pH=8.0) was added, the mixture was allowed to stand at 37℃for 2 hours, and 12000g was centrifuged for 5 minutes, and the supernatant was discarded. The pellet was subjected to plasmid extraction as described in the miniplasmid kit and the extracted plasmid was observed by electrophoresis.
After plasmid pNZ8149 is subjected to double-enzyme tangential, seamless Cloning Kit (seamless cloning kit) is adopted to be added into a reaction system together with a target gene DI-DII, and the subsequent electrotransformation reaction is carried out after the reaction is carried out for 15min at 50 ℃. Culturing for 16-18h with EM culture medium with lactose as unique carbon source, and picking out suspected positive colony as shown in figure 4, wherein the left side of figure 4 is the result after electric conversion of pNZ8149-DI-DII, and the right side of figure is the result after electric conversion of pNZ 8149.
2.4 Cleavage and recovery of pNZ8149 plasmid
The pNZ8149 plasmid was digested with Nco I and Sac I overnight at 37℃with the specific digestion system shown in tables 2-3, and then water-bath was carried out at 37℃for 2h. And (3) observing the electrophoresis of the digested pNZ8149, and carrying out gel cutting recovery for standby after the complete digestion is determined.
Tables 2-3 double cleavage reaction System
2.5 Seamless cloning of the fragment of interest and the pNZ8149 plasmid
The target gene and the digested target fragment pNZ8149 plasmid are subjected to seamless cloning connection reaction by using Biyun Tian Seamless Cloning Kit (seamless cloning kit), and the reaction system is placed in a temperature of 50 ℃ for 15min for incubation, so that transformation can be performed. The specific connection reaction system is shown in tables 2 to 4.
Tables 2 to 4 connection reaction system
3) Preparation of NZ3900 competent bacteria
(1) Streaking lactobacillus NZ3900 on G-M17 plate, and culturing at 30deg.C overnight
(2) The following day, the monoclonal was picked up in G-SGM17 medium and incubated overnight at 30 ℃.
(3) Thirdly, transferring the lactobacillus NZ3900 culture solution into G-SGM17 according to the proportion of 1:10, and standing and culturing at 30 ℃ overnight;
(4) The lactobacillus culture solution NZ3900 is amplified and cultured into G-SGM17 according to the ratio of 1:8, and then is subjected to stationary culture at 30 ℃ until OD 600 = 0.2-0.3, and is centrifuged at 8000rpm and 4 ℃ for 10min, so as to collect the thalli.
(5) Re-suspending the thallus with 10-20mlEPB, centrifuging at 8000rpm and 4 deg.c for 10min, and collecting thallus;
(6) Re-suspending the cells with 50ml EPB+EDTA for 15min, centrifuging to collect the cells, re-suspending the cells with 5-10mlEPB, centrifuging to collect the cells, concentrating the re-suspended cells with EPB at a ratio of 100:1 of the volume of the cells finally expanded and cultured, and placing on ice.
(7) Then, the thalli are packaged rapidly, 40ul each is stored at-80 ℃.
4) Plasmid electrotransfer lactic acid lactococcus
40Ul of lactic acid bacteria NZ3900 competent bacteria were placed on ice for 2-3min, then 4ul (empty plasmid 1 ul) of the ligation product was added, and the mixture was allowed to stand on ice for 15min, transferred into a 0.2mm electric cup (pre-cooling of the electric cup in advance), and subjected to electric transfer in the following manner: the fungi, shs mode, shows a shock time after shock, with an optimal shock time of 4.5ms. And after the electrotransport process is finished, rapidly resuscitating with 1ml of electrotransport resuscitation fluid prepared by ice. Ice pre-treatment for 5min, standing at 30deg.C for 1.5h, centrifuging in a centrifuge tube, removing part of supernatant in a super clean bench, re-suspending thallus, collecting 50ul coated plate, culturing at 30deg.C for 18h, and culturing and identifying after monoclonal is obtained.
5) Identification of recombinant plasmids
5.1 Plasmid identification
The suspected positive clone obtained in the last step is selected and the monoclonal bacteria are placed in an EM culture medium for overnight culture at 30 ℃, the bacterial liquid is subjected to plasmid extraction (the method is the same as that of the plasmid extraction of pNZ 8149) in the next day, and preliminary judgment is carried out through the size of the plasmid and the size comparison with the size of the pNZ 8149.
After positive colony culture is selected, recombinant plasmids are extracted, comparison and gel running are carried out on the recombinant plasmids and the empty plasmids, and the difference of the sizes of the strips can be seen from the graph in FIG. 5, M1:0.2-1.2KD DNA Marker;1: pNZ8149,2: pNZ8149-DI-DII, then the suspected positive plasmid was sequenced and the success of the recombinant plasmid construction was determined.
5.2 PCR identification
PCR was performed using the plasmid of the positive clone as a template (the system and reaction conditions are the same as above). After completion of PCR, electrophoresis was performed for observation.
5.3 Sequencing identification
The extracted plasmid was submitted to Nanjing Jin Weizhi Biotechnology Inc. for sequencing and alignment with the original gene sequence by DNAStar software.
6) Expression and analysis of exogenous target protein of recombinant lactococcus lactis
6.1 Inducible expression of exogenous target protein of recombinant lactococcus lactis and protein extraction
Protein induction expression and protein preparation: marking and resuscitating recombinant lactobacillus containing different target fragments on an EM (effective microorganism) plate; selecting a monoclonal strain, standing and culturing at 30 ℃ overnight, and mixing activated lactobacillus according to the following formula 1:50 was transferred to fresh EM, and when OD 600 =0.4-0.5, nisin was added to induce at a final concentration of 1ng/ml, and the mixture was allowed to stand at 30℃for 3 hours. After the induction, 2ml of the bacterial liquid was collected, the bacterial cells and the supernatant were centrifuged, the pellet was suspended in an appropriate amount of ice-cooled 10mM Tris-hcl (pH=8.5), centrifuged at 8000rpm/min, and the supernatant was discarded. Repeated 2 times, resuspended in 140. Mu.L of 10mM Tris-hcl (pH=8.5) containing 1mg/mL lysozyme, reacted at 37℃for 1h, centrifuged, the supernatant was discarded, the cells were lysed by mixing with 30. Mu.L of 20wt% SDS, 70. Mu.L of 10mM Tris-hcl (pH=8.5) was added, 50u15 Xprotein loading buffer was added, and the mixture was stirred with water for 10min. Then, the sample was cooled in an ice box, 10. Mu.L of the sample was taken as a sample, and the remainder was kept at-80 ℃.
Protein extraction: after the induction is finished, the thalli are centrifugally separated, washed for 2 times by PBS, and concentrated and resuspended by 1:10 volume; the cell ultrasonic disruption instrument is used for carrying out ultrasonic disruption on thalli, and the specific procedures are as follows: the power is 40W, the ultrasonic wave is 5s, the interval is 7s, and the ultrasonic wave is 20min. The sonicated sample was centrifuged at 8000rpm,4℃for 10min, the supernatant and pellet were separated, and pellet was prepared using 1:10 proportion PBS, mixing with supernatant with small amount of protein Loading Buffer, boiling for 10min, and storing at-80deg.C.
7) SDS-PAGE and Western blot detection of exogenous target protein
SDS-PAGE: taking a standard pre-dyed protein Maker as a control, taking the 20ul of prepared target protein sample and pNZ8149 plasmid control for SDS-PAGE detection (5 wt% of concentrated gel and 12wt% of separation gel), dyeing with coomassie brilliant blue for 1h after electrophoresis, adding a decolorizing solution for decolorizing for 2h after the dyeing reaction is completed, and observing the result.
The recombinant expression plasmid pNZ8149-DI-DII was induced in lactic acid bacteria NZ3900 by Nisin inducer, stained with Coomassie Brilliant blue by SDS-PAGE and decolorized, and the protein band was seen at 35KD, see FIG. 6, M:10-150KD protein marker;1: pNZ8149-DI-DII;2: pNZ8149, but is not clearly distinguishable from the control group, and therefore requires further experimental verification.
Western blot: taking 20ul of prepared target protein sample and pNZ8149 plasmid contrast to carry out SDS-PAGE, transferring the protein onto a PVDF membrane by using a wet transfer method, sealing 10mih by using a rapid sealing liquid at room temperature, washing, taking a murine E protein polyclonal antiserum diluted 1000 times by PBS as a primary antibody to incubate for 1h, washing, taking HRP-marked goat anti-mouse IgG as a secondary antibody to incubate for 1h, washing, and using ECL color development to carry out color development observation on a gel imaging system.
Recombinant expression plasmid pNZ8149-DI-DII was induced in lactic acid bacteria NZ3900 by Nisin inducer and analyzed by western-blot, see FIG. 71 at 35 KD: pNZ8149-DI-DII;2: the pNZ8149,1 has a distinct western blotting band, which shows that only the recombinant plasmid pNZ8149-DI-DII expression product has immunoreactivity for binding with tambusu E protein antisera after the expression of lactococcus lactis, indicating that only pNZ8149-DI-DII expresses the protein.
8) Optimization of expression condition of exogenous target protein of recombinant lactococcus lactis
In order to obtain a better expression effect, the target protein optimizes the expression condition through a 3-factor 3-level orthogonal experiment, and the specific experimental combination is as follows:
TABLE 2-5 Induction Condition optimization
After the recombinant lactic acid bacteria NZ3900-pNZ8149-DI-DII were optimally expressed, the Western blot bands in 3 were thickest and the expression level was highest as shown in FIG. 8. The optimal protein induction expression condition is that the concentration of an inducer is lng/ml, the OD 600 nm value is 0.5, and the induction time is 5h.
1: Nisin: lng/ml, induction time: 2h, OD600nm:0.3,2: nisin:1ng/ml, induction time: 3h, OD 600 nm:0.4,3: nisin:1ng/ml, induction time: 5h, OD 600 nm:0.5,4: nisin:10ng/ml, induction time: 2h, OD 600 nm:0.4,5: nisin:10ng/ml, induction time: 3h, OD 600 nm:0.5,6: nisin:10ng/ml, induction time: 5h, OD 600 nm:0.3,7: nisin:100ng/ml, induction time: 2h, OD 600 m:0.5,8: nisin:100ng/ml, induction time: 3h, OD 600 nm:0.3,9: nisin:100ng/ml, induction time: 2h, OD 600 nm:0.4
9) Recombinant lactococcus lactis NZ3900-pNZ8149-DI-DII protein expression position detection
After the recombinant lactobacillus is induced by the optimal induction expression condition, carrying out ultrasonic on ice, taking the sediment and the supernatant to carry out western-blot experiment identification, and as shown in figure 9 (1: supernatant after PBS (PBS) resuspension ultrasonic disruption 2: supernatant after PBS resuspension ultrasonic disruption), both the soluble supernatant and the sediment are expressed, and the sediment expression quantity is high.
In summary, since the neutralizing antibody is generated after the organism is infected by the virus, the neutralizing antibody has the effect of preventing the organism from re-infecting the same virus, thereby achieving the effect of preventing the virus infection. Thus, during the development of a vaccine, whether the vaccine can cause the body to produce the corresponding neutralizing antibodies is the standard for evaluating the effectiveness of the vaccine. The lactococcus lactis live vector selected in the experiment can carry exogenous protein to reach the intestinal tract, and stimulate the intestinal tract to generate neutralizing antibodies aiming at the exogenous protein, so that the immune effect is achieved. The DI-DII fragment of E gene is selected as exogenous expression protein mainly because the region has epitope which can stimulate the generation of neutralizing antibody, domainIII protein and DI-DII protein have difference in structure, domainIII protein is similar to 1gG structure, but practical application protein is difficult to express, and because the structure is similar to lgG and is complex, the DI-DII protein is difficult to express in lactococcus lactis carrier, therefore, the DI-DII protein accords with the selection condition of novel live carrier subunit vaccine epitope unit.
The carrier selected by the application is food-grade, does not carry any resistance gene, and meets the requirements. The DI-DII structure has a hydrophilic region, so that the protein structure is not too complex, and the DI-DII structure has the characteristics of higher expression quantity and solubility when actually expressing exogenous proteins, and is suitable for practical application.
The E gene DI-DII region protein is used as a recombinant lactococcus lactis oral carrier vaccine, and is the first attempt of duck tembusu virus vaccine development. And a series of experimental verification results show that the expression quantity of the exogenous protein is very considerable and the cost is low, thereby providing data support for the vaccine immune effect research under the actual production condition.
Claims (8)
1. The construction method of the food-grade recombinant duck tembusu virus truncated E gene lactococcus lactis vector is characterized by comprising the following steps in sequence:
1) Optimized synthesis of full-length E gene fragments
The gene sequence of a duck Tembusu virus JM strain with the GenBank accession number of JN811559 is subjected to codon optimization according to host bacterium MG1363, the full-length sequence 1502bp of an E gene is synthesized, two restriction sites of NcoI and SacI are inserted into two endpoints of the full-length sequence of the E gene and are filled into pUC57 plasmids, the recombinant plasmid is transformed into Top10 clone strains, and the recombinant plasmid is named pUC57-E;
2) Construction of NZ3900-pNZ8149-DI-DII recombinant expression vector
2.1 Primer design)
Designing a primer for amplifying the DI-DII fragment, wherein the nucleotide sequence of the DI-DII is shown in SEQ ID NO: as shown in figure 1, the number of the components,
The upstream primer sequence of the DI-DII fragment is as follows: F-5'-TAAGGAGGCACTCACATGTTTTCATGTTTGGGTATGCAAAAT-3';
the sequence of the downstream primer of the DI-DII fragment is as follows: R-5'-GTTCAAAGAAAGCTTGAGCTCATACCTTTTAACTTTAAACC-3';
2.2 Fragments of interest) acquisition of (a)
Amplifying the target DI-DII fragment by using the pUC57-E of the step 1) as a template for PCR amplification reaction;
2.3 Preparing lactococcus lactis NZ3900 competent bacteria, and preserving at-80 ℃ for later use;
2.4 Extraction of pNZ8149 plasmid
Electrotransferring the plasmid pNZ8149 into lactococcus lactis NZ3900 competent bacteria, picking positive clone and culturing, and extracting the plasmid pNZ 8149;
2.5 Cleavage and recovery of pNZ8149 plasmid
Carrying out double enzyme digestion on the pNZ8149 plasmid by using NcoI and SacI, and recovering a 2500bp vector fragment to obtain a linearized pNZ8149 vector;
2.6 Seamless cloning of the fragment of interest and the pNZ8149 plasmid
Carrying out homologous recombination connection reaction on the target DI-DII fragment after glue recovery and the pNZ8149 plasmid, and incubating a reaction system at 50 ℃ for 15min to obtain a connection product so as to carry out electric conversion;
2.7 Recombinant plasmid electrotransfer lactococcus lactis competent bacteria
Taking 40 mu L of lactococcus lactis NZ3900 competent bacteria, placing the bacteria on ice for 2-3min, adding 4 mu L of the connection product, standing on ice for 15min, transferring into a 0.2mm electric rotating cup for electric rotating, and rapidly resuscitating by using 1mL of ice pre-cooled electric rotating resuscitating liquid after electric rotating is finished; pre-cooling with ice for 5min, standing at 30 ℃ for 1.5h, transferring into a centrifuge tube, centrifuging, discarding the supernatant in a super clean bench, re-suspending thalli, taking a 50 mu L coating plate, culturing for 18h at 30 ℃ in an inverted manner, and selecting monoclonal bacteria for culture identification after the monoclonal bacteria grow out;
3) Identification of recombinant plasmids
3.1 Plasmid identification
Culturing the grown suspected positive monoclonal bacteria in an EM liquid culture medium at 30 ℃ overnight, extracting plasmids from the bacterial liquid the next day, and performing preliminary judgment by comparing the size of the recombinant plasmids with that of pNZ 8149;
3.2 PCR identification
Taking plasmids extracted from suspected positive monoclonal bacteria as templates, and carrying out PCR identification by using the primers; performing electrophoresis for observation;
3.3 Sequencing identification
Sequencing the extracted plasmid, and comparing the extracted plasmid with the optimized E gene sequence through DNAStar software;
4) Expression of exogenous target protein of recombinant lactococcus lactis
Selecting positive monoclonal bacteria, standing and culturing at 30 ℃ overnight, and mixing the activated positive monoclonal bacteria according to the following formula 1:50 transferring into fresh EM culture medium, adding Nisin with a final concentration of 1ng/mL for induction when OD 600 = 0.4-0.5, and standing at 30deg.C for 5h; after the induction, 2mL of bacterial liquid is taken for centrifugal separation of thalli and supernatant, a proper amount of ice-cooled 10mM Tris-HCl is used for precipitation, suspended cells with PH=8.5 are centrifugated at 8000rpm/min, and the supernatant is discarded; repeating for 2 times, re-suspending with 140 mu L of 10mM Tris-HCl containing 1mg/mL lysozyme, wherein the pH=8.5, reacting for 1h at 37 ℃ in an incubator, centrifuging, discarding the supernatant, adding 30 mu L of 20wt% SDS, uniformly mixing and cracking thalli, adding 70 mu L of 10mM Tris-HCl, wherein the pH=8.5, adding 50 mu L of 5X protein loading buffer solution, blowing and uniformly mixing, and then carrying out boiling water bath for 10min; and then placing the sample in an ice box for cooling, taking 10 mu L as a sample loading quantity, and storing the rest at-80 ℃.
2. The method for constructing the food-grade recombinant duck tembusu virus truncated E gene lactococcus lactis vector according to claim 1, wherein the PCR amplification reaction system comprises the following components in parts by volume: 2 Xbuffer 25 [ mu ] L; dNTP 10 [ mu ] L; the upstream primer 1[ mu ] L; the downstream primer 1[ mu ] L; KODFx 1[ mu ] L; ddH 2 O11 [ mu ] L; pUC 57-E1 [ mu ] L; the total volume is 50 mu L; the reaction conditions are as follows: 94 ℃,2min,98 ℃,10s,56 ℃,30s,68 ℃,30s,35 cycles, and 68 ℃ for 10min.
3. The method for constructing a food-grade recombinant duck tembusu virus truncated E gene lactococcus lactis vector according to claim 1, wherein the plasmid extraction is prepared by the following steps: lactococcus lactis NZ3900 strain containing pNZ8149 plasmid is subjected to standing overnight culture at 30 ℃ with an EM culture medium, 5mL of bacterial liquid is taken for the next day, 12000g is centrifuged for 1min, and the supernatant is discarded; washing 2-3 times with 10mM Tris-HCl, pH=8.5, centrifuging 12000g for 1min, and discarding the supernatant; 1mL of lysozyme with pH=8.0 and 20mg/mL is added, the mixture is kept stand at 37 ℃ for 2 hours, 12000g is centrifuged for 5min, and the supernatant is discarded; the pellet was subjected to plasmid extraction and electrophoresis observation according to the instructions of the miniplasmid kit.
4. The method for constructing a truncated E gene lactococcus lactis vector of a food-grade recombinant duck tembusu virus according to claim 1, wherein the enzyme digestion system in step 2.5) comprises the following components in parts by volume: pNZ8149 30 [ mu ] L; ncoI 1 [ mu ] L; sacI 1 [ mu ] L;10 Xbuffer 5 [ mu ] L; ddH 2, 13 [ mu ] L; totaling 50 μl.
5. The method for constructing a truncated E gene lactococcus lactis vector of a food-grade recombinant duck tembusu virus according to claim 1, wherein the ligation reaction system in step 2.6) comprises the following components in volume: pNZ8149 [ mu ] L; the destination DI-DII fragment 3 [ mu ] L;2XSeamLessCloningMix 10 [ mu ] L; ddH 2 06 [ mu ] L; totaling 20 μl.
6. The method for constructing a truncated E gene lactococcus lactis vector of a food-grade recombinant duck tembusu virus according to claim 1, wherein the competent bacterium of the lactococcus lactis NZ3900 is prepared by the following steps:
(1) Lactococcus lactis NZ3900 was streaked onto G-M17 plates and incubated overnight at 30 ℃;
(2) The next day, the lactococcus lactis NZ3900 monoclonal bacteria obtained in the step (1) are selected and cultured in G-SGM17 culture solution at 30 ℃ overnight;
(3) On the third day, the lactococcus lactis NZ3900 obtained in the step (2) is transferred into G-SGM17 according to the proportion of 1:10, and the mixture is subjected to standing culture at 30 ℃ overnight;
(4) Lactococcus lactis NZ3900 is subjected to expansion culture into G-SGM17 according to the ratio of 1:8, standing culture is performed at 30 ℃ until OD 600 = 0.2-0.3, then centrifugation is performed at 8000rpm and at 4 ℃ for 10min, and thalli are collected;
(5) Re-suspending the cells with 10-20mL EPB, centrifuging at a speed of 8000rpm and a temperature of 4 ℃ for 10min, and collecting the cells;
(6) The cells were resuspended with 50mL of EPB+EDTA, pre-frozen for 15min, then centrifuged to collect the cells, resuspended with 5-10mL of EPB, centrifuged to collect the cells, and finally expanded with EPB according to the final cell volume of 100:1, concentrating and resuspending thalli in proportion, and placing on ice;
(7) And then the thalli are rapidly packaged, and each thalli is stored at the temperature of minus 80 ℃ and 40 mu L.
7. The method for constructing a truncated E gene lactococcus lactis vector of a food-grade recombinant duck tembusu virus according to claim 1, further comprising detecting an exogenous target protein by SDS-PAGE and Western blot;
the SDS-PAGE detection method comprises the following steps: performing SDS-PAGE detection on the prepared target protein sample, dyeing with coomassie brilliant blue for 1h after electrophoresis, adding a decolorizing solution for decolorizing for 2h after the dyeing reaction is completed, and observing the result;
The SDS-PAGE detection adopts 5wt% of concentrated gel and 12wt% of separation gel;
The Western blot detection method comprises the following steps: after SDS-PAGE is carried out on the proteins, protein gel is cut off according to the expected size of the target proteins, then the proteins are transferred onto PVDF membrane by a wet transfer method, the PVDF membrane is sealed for 10min at room temperature by a rapid sealing liquid after cleaning, the polyclonal antiserum of the murine E protein is used as a primary antibody for incubation of 1h after cleaning, goat anti-mouse IgG marked by HRP is used as a secondary antibody for incubation of 1h after cleaning, and the result is observed under a thin-layer gel scanner by ECL chemiluminescent color developing liquid after cleaning.
8. Use of a recombinant food-grade duck tembusu virus truncated E gene lactococcus lactis vector constructed according to claim 1 for producing active duck tembusu virus truncated E gene domainI-domainII fragment protein, characterized in that the production conditions are: the culture was allowed to stand at 30℃for 5 hours under Nisin at a final concentration of 1 ng/mL.
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| CN107312782A (en) * | 2017-07-28 | 2017-11-03 | 浙江省农业科学院 | Duck tembusu virus E protein truncate gene, restructuring duck plague virus and construction method and application |
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