CN117660276A - Recombinant lactococcus lactis strain, construction method and application thereof - Google Patents
Recombinant lactococcus lactis strain, construction method and application thereof Download PDFInfo
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Abstract
The invention relates to the fields of molecular biotechnology and microorganisms, in particular to a recombinant lactococcus lactis strain, a construction method and application thereof, which are used for solving the defects of pathogenic variation, attenuated live vaccine immune failure and the like of the current commercial vaccine in the epidemic prevention and control aspect. The strain comprises fusion genes of type 1 and type 3 duck hepatitis A virus VP1 genes, the fusion genes are introduced into lactic acid emulsion balls through plasmid vectors, and the fusion genes have DNA sequences shown in SEQ No. 1. Experimental results show that the lactococcus lactis for expressing the type 1 and type 3 duck hepatitis A virus VP1 fusion proteins has good immunoprotection on duckling infected by DHAV-1 single infection, DHAV-3 single infection and DHAV-1 and DHAV-3 mixed infection.
Description
Technical Field
The invention relates to the technical field of unmanned underwater vehicles, in particular to a construction method of a recombinant lactococcus lactis strain.
Background
Duck viral hepatitis (Duck viral hepatitis, DVH) is mainly caused by duck hepatitis A virus (Duck hepatitis Avirus, DHAV) infection, and is one of the most important virulent infectious diseases endangering the duck industry. The disease is characterized by acute liver injury, and the disease mortality rate of duckling is up to 90%. Although vaccines and egg yolk antibodies are used for preventing and treating the disease, the failure of immunity happens, which seriously hampers the healthy development of duck raising industry in China. There are three serotypes of duck hepatitis A Virus, of which types 1 and 3 are the dominant serotypes in our country. The clinical symptoms and pathological changes of ducklings infected by DHAV-1 and DHAV-3 are very similar, and the mixed infection phenomenon of the two is generated in the production, thus adding difficulty to the prevention and treatment of the disease.
The current commercial vaccine has the defects of pathogen variation, attenuated live vaccine immune failure and the like in the epidemic prevention and control aspect.
The microecological preparation can be used for immunization through drinking water feeding, the method is considered as an acceptable immunization method, the administration route is convenient and safe, and the large-group immunization can be rapidly developed at the same time, so that the stress on organisms can be avoided, the labor force can be saved, the immunization cost can be reduced, and the organisms are protected against pathogen attack.
Disclosure of Invention
The invention aims to provide a recombinant lactococcus lactis, a construction method and application thereof, wherein the strain comprises fusion genes of type 1 and type 3 duck hepatitis A virus VP1 genes, and can anchor and express exogenous VP1 fusion proteins on the cell walls of the lactobacilli, and the fusion genes are introduced into the lactococcus lactis through plasmid vectors.
The invention aims to provide a recombinant lactococcus lactis strain, which is preserved in China general microbiological culture collection center (CGMCC) No.26965lactis-DHAV-1/3VP1 in the year 3 and the month 30 of 2023.
The coccus contains fusion genes of type 1 and 3 duck hepatitis A virus VP1 genes, and can anchor and express exogenous VP1 fusion proteins on the cell wall of lactic acid bacteria, and the fusion genes are introduced into the lactic acid lactococcus through a plasmid vector.
Another object of the present invention is to provide a method for constructing a recombinant lactococcus lactis strain, comprising the steps of:
step 1: extracting genome RNA of type 1 and type 3 duck hepatitis A viruses, respectively carrying out reverse transcription, taking the obtained cDNA as a template, designing primers to respectively amplify two pathogenic VP1 gene fragments, connecting to form VP1 (1, 3) through homologous recombination, and amplifying by a specific primer to ensure that two ends of the sequence respectively contain pgsA' and eGPF gene homology arms;
step 2: extracting bacillus subtilis genome DNA, designing a primer for amplification to obtain pgsA' fragments with both ends respectively containing Usp45 and VP1 (1, 3) gene homology arms;
step 3: designing a primer to amplify by taking a T7g10L-Usp45-VP1-eGFP-pMG36e plasmid as a template to obtain a T7g10L-Usp45 fragment with both ends containing a pMG36e vector and pgsA' homology arms;
step 4: designing primers to amplify eGFP fragments containing VP1 genes and pMG36e homology arms at two ends by taking pEGFP-C3 plasmid as a template;
step 5: connecting the gene fragments obtained in the steps 1, 2, 3 and 4 through homologous recombination to obtain a fusion fragment T7g10L-Usp45-pgsA' -1VP1-3VP1-eGFP;
step 6: the fusion fragment and a linearization vector pMG36e are connected through homologous recombination to obtain a recombinant plasmid Usp45-pgsA' -1VP1-3VP1-eGFP-pMG36e;
step 7: transforming the recombinant plasmid obtained in the step 6 into escherichia coli DH5 alpha, screening positive transformants by erythromycin resistance, picking single colony for PCR verification and sequencing, and culturing the correct recombinant single colony at 37 ℃ to obtain a cloning plasmid;
step 8: and (3) electrically transferring the recombinant plasmid obtained in the step (7) into lactococcus lactis MG1363, screening positive strains by using erythromycin resistance, selecting single colony PCR for verification, preserving the correct strains, and storing the strains at-80 ℃.
Furthermore, the fusion gene in the step 5 has a DNA sequence shown in SEQ No.1, and has an anchoring sequence pgsA' for improving the expression rate of the exogenous protein on the surface of the lactobacillus, and a green fluorescent protein eGFP sequence capable of directly monitoring the expression and the localization mode of the target protein.
Still further, the lactococcus lactis is lactococcus lactis MG1363.
Another object of the invention is to provide a duck viral hepatitis immune microecological preparation comprising the recombinant lactococcus lactis as described above.
Further, the concentration of recombinant lactococcus lactis thallus in the immune microecological preparation is as follows: 5.0X10 8 cfu/mL. The dosage of administration is preferably 1.0mL.
Still further, the immune microecological formulation is a vaccine administered by oral gavage.
The invention also aims to provide a preparation method of the duck viral hepatitis immune microecological preparation, which specifically comprises the following steps: respectively amplifying VP1 genes from the duck hepatitis A virus genome type 1 and the duck hepatitis A virus genome type 3, and simultaneously amplifying a signal peptide Usp45 sequence and a pgsA' sequence; then, the DHAV-1VP1 and DHAV-3VP1 genes are inserted into a pMG36e vector in series through a homologous recombination method, an anchor sequence pgsA' is introduced, a recombinant plasmid vector carrying DHAV-1VP1 and DHAV-3VP1 fusion genes is constructed, the plasmid vector is introduced into lactococcus lactis MG1363, recombinant lactococcus lactis is constructed, and the recombinant lactococcus lactis containing the DHAV-VP1 (1, 3) fusion genes is utilized to prepare the duck viral hepatitis immune microecologics.
Compared with the prior art, the invention has the following advantages:
the invention successfully constructs the lactococcus lactis strain for expressing the type 1 and type 3 duck hepatitis A virus VP1 fusion proteins, and the strain can enable the fusion proteins to be anchored and expressed on cell walls. Animal virus attack protection tests show that the lactococcus lactis expressing the type 1 and type 3 duck hepatitis A virus VP1 fusion protein has a relative protection rate of 64.71% for the DHAV-1 single-infected duckling, 61.54% for the DHAV-3 single-infected duckling and 57.89% for the DHAV-1 and DHAV-3 mixed-infected duckling, and has a good immune protection effect.
Drawings
FIG. 1 is a diagram showing construction of recombinant plasmid in E.coli DH 5. Alpha
FIG. 2 shows fluorescence microscopy results, wherein A is the bright field of recombinant lactococcus lactis; b is a fluorescent field of recombinant lactococcus lactis; c is a bright field containing pMG36e lactococcus lactis; d is the fluorescence field of pMG36e lactococcus lactis.
FIG. 3 is an index measurement during fermentation of lactococcus lactis; wherein A is the change of the number of living bacteria along with the fermentation time; b is the change of the lactococcus lactis OD 600nm with the change of fermentation time.
FIG. 4 shows the result of Westernblot analysis for induced expression of DHAV-VP1 (1, 3). Wherein M: protein molecular mass standard; +: lactococcus lactis MG1363/VP1 (1, 3); -: lactococcus lactis MG1363/pMG36e. Wherein A uses monoclonal antibody 4F8 as primary antibody; b uses monoclonal antibody eGFP-Tag as primary antibody.
Fig. 5 is a graph showing the number of ducklings survived natural infection with DHAV.
FIG. 6 is a graph of immune levels in a duckling serum or intestinal tract sample; A. b, C, D, E are graphs of changes in the levels of IgG, sIgA, IL-4 and IFN-gamma, respectively.
Detailed Description
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. Further, it is understood that various changes and modifications may be made by those skilled in the art after reading the teachings of the present invention, and such equivalents are intended to fall within the scope of the claims appended hereto.
Duck hepatitis A Virus is the only member of the genus avian hepacivirus of the family picornaviridae, and has three serotypes: DHAV-1, DHAV-2 and DHAV-3, wherein DHAV-1 and DHAV-3 are dominant serotypes of our country. The DHAV genome has a structure of 5'UTR-VP0-VP3-VP1-2A (2A 1-2A2-2A 3) -2B-2C-3A-3B-3C-3D-3' UTR-poly (A). Among the encoded products, VP1 protein contains neutralizing epitopes, which are effective in inducing the production of protective antibodies, and is considered as the most important immunogenic protein of the virus. Therefore, we amplify VP1 genes of DHAV-1 and DHAV-3, respectively, and then tandem them for gene fusion expression.
The lactococcus lactis is preserved in China general microbiological culture collection center with a preservation number of CGMCC No.26965 and a preservation time of 2023, 3 and 30 days. The address is the post code 100101 of the institute of microbiology of national academy of sciences, no. 3, north chen west way 1, the region of the morning sun in Beijing city.
The recombinant plasmid vector carrying the DHAV-1VP1 and DHAV-3VP1 fusion genes is constructed by inserting the DHAV-1VP1 and DHAV-3VP1 genes into a pMG36e vector in series and introducing an anchor sequence pgsA', recombinant lactococcus lactis is constructed by introducing the plasmid vector into lactococcus lactis MG1363, and the recombinant lactococcus lactis containing the DHAV-VP1 (1, 3) fusion genes is used for preparing the duck viral hepatitis immune microecological preparation.
For a further understanding of the technical solutions of the present application, the following examples are now provided:
example 1:
construction of recombinant plasmid carrying DHAV-VP1 (1, 3) fusion Gene
VP1 gene is amplified from LY0801 strain and SD1201 strain of type 3 duck hepatitis A virus, and simultaneously signal peptide Usp45 sequence and pgsA' sequence are amplified. The pMG36e plasmid is used as a carrier plasmid to construct a recombinant plasmid carrying the DHAV-VP1 (1, 3) fusion gene.
The PCR primer sequences for amplifying VP1 gene, usp45 gene, eGFP gene and pgsA' gene are shown in the following table 1, wherein eGFP label is added at the tail end of the target gene primer, and a universal primer of pMG36e is selected as an identification primer.
TABLE 1 PCR primer sequences for amplifying VP1 Gene, usp45 Gene, eGFP Gene and pgsA' Gene
Using plasmid T7g10L-Usp45-VP1-eGFP-pMG36e as template, amplifying T7g10L-Usp45 fragment with primer 1F/1R; amplifying the fragment pgsA' by using the bacillus genome as a template and using a primer 2F/2R; the plasmid pR-DHAV-1 was used as a template to amplify the DHAV-1/VP1 fragment with primer 3F/3R; the plasmid pR-DHAV-3 was used as a template to amplify the DHAV-3/VP1 fragment with primer 4F/4R; fragment eGFP was amplified with primer 5F/5R using plasmid pEGFP-3C as template.
T7g10L-Usp45, pgsA ',1VP1,3VP1, eGFP were fused together to obtain a 2955bp fusion gene T7g10L-Usp45-pgsA' -1VP1-3VP1-eGFP with a pMG36e homology arm. The fusion flow is shown in fig. 1.
The plasmid and fusion gene were double digested with restriction enzymes Xba I and Hind III, and the final 2911bp fragment was inserted into plasmid pMG36e. The recombinant plasmid was transformed into E.coli DH 5. Alpha. Competent cells.
Competent cells of lactococcus lactis MG1363 were prepared in advance. Plasmid is subjected to
Usp45-pgsA' -1VP1-3VP1-eGFP-pMG36e was transferred to competent cells by electroporation and the cells were cultured in GM17 agar medium containing erythromycin at a concentration of 1.0. Mu.g/mL. The positive plasmid screened was transformed into lactococcus lactis MG1363 twice. The positive recombinant MG1363 strain contains T7g10L-Usp45-pgsA' -1VP1-3VP1-eGFP-pMG36e, more named MG13631+3VP1. Lactococcus lactis MG1363/pMG36e was used as a negative control. Recombinant lactococcus lactis was observed under Nikon-orthographic fluorescence microscope 55i as shown in FIG. 2.
Example 2: identification of recombinant lactococcus lactis fusion Gene expression
Recombinant lactobacillus MG1363/VP1 (1, 3) and MG1363/pMG36e are firstly added into GM17 culture medium without erythromycin, and are cultured for 18 hours at 37 ℃, OD 600nm detection is carried out on the culture medium every 2 hours, and the number of living bacteria is calculated, wherein the number of living bacteria and the change trend of OD 600nm are shown in figure 3. Bacterial suspension was centrifuged at 5000rpm for 10 minutes, and bacterial pellet and supernatant were separated. The bacterial pellet was resuspended in TES solution, allowed to stand at 37℃for 30 minutes, centrifuged again, the supernatant removed and resuspended in TE buffer, centrifuged at 5000rpm for 10 minutes, and the pellet resuspended in ultrapure water and freeze-thawed repeatedly 5 times. Centrifugation was then performed at 4℃and the pellet was resuspended in PBS. Finally, separating target protein by utilizing Anti-GFP magnetic beads. Western blot assays are performed on the isolated target protein to detect the presence of the target protein on the cell wall. The target fusion protein has a molecular weight theoretical value of 72kDa and a result of Western blot measurement is shown in FIG. 4, wherein the monoclonal antibodies of the monoclonal antibodies 4F8 and the eGFP-Tag are respectively used as primary antibodies, the goat anti-mouse IgG coupled with HRP is used as secondary antibodies.
Example 3: immune protection effect of recombinant lactococcus lactis
277 daily-old cherries Gu Chuya were purchased, and 5 serum and intestinal samples were collected at random.
Ducklings were divided into two groups, designated as experimental and control groups. The experimental and control groups were divided into 4 groups, ABCD and EFGH, respectively. The duckling of the experimental group is orally fed with 1mL of immune microecological preparation (the concentration of the recombinant lactococcus lactis is 5.0X108 cfu.mL < -1 >) at the ages of 2, 4, 10 and 14 days, and the duckling of the control group is fed with the same amount of sterile water. 12 ducklings were randomly collected from group G at 15 days of age, 1mL of DHAV was injected, and 2 challenged ducklings were respectively placed into the ABCEFG group to simulate natural infection. Group A and group E were placed into ducklings infected with DHAV-1 and designated as DHAV-1 experimental and DHAV-1 control groups, respectively. Group B and group F were placed into ducklings infected with DHAV-3, designated DHAV-3 experimental and DHAV-3 control groups, respectively. Group C and group G were placed in ducklings simultaneously infected with DHAV-1 and DHAV-3, designated DHAV-1+3 experimental and DHAV-1+3 control groups, respectively. Starting at 15 days of age, the dead ducklings were recorded and a survival curve was drawn, and 3d after infection was found, ducklings of the control group were acutely dead, and ducklings fed lactococcus lactis MG1363/1+3VP1 orally did not die until 4d after infection. The ducks of the DHAV-1 experimental group died at 20 days old and 21 days old, and the ducks of the DHAV-1 control group died continuously and rapidly at 17-21 days old. The DHAV-3 experimental group showed small deaths at 20, 21 and 22 days of age. The DHAV-3 control group ducks showed massive death at 18-21 days of age. The ducks of the DHAV-1 and DHAV-3 experimental groups showed small amounts of death at 20-23 days of age, while the ducks of the control group died rapidly at 17-20 days of age, and the death at 21-24 days of age slowed down, as shown in FIG. 5. According to the formula: relative Percent Survival (RPS) = (experimental group mortality/control mortality) ×100%, and the results of the animal challenge protection experimental immunization effect were counted as shown in table two.
Immune effect of exterior two animals challenge protection experiment
After immunization, 5 ducklings were randomly collected at 4, 7, 10, 14, 17, 20, 22 and 24 days of age in each group, and serum and duodenal samples were collected. Repeatedly washing the duodenal sample with PBS buffer solution, oscillating the washing solution on an oscillator for 1h, centrifuging at 12000rpm for 10min at 4 ℃ in a centrifuge, sucking the supernatant, adding BSA to a final concentration of 0.1%, and obtaining the small intestinal mucosa liquid sample. Diluting the purified protein coating, adding the diluted protein coating into a 96-well plate, adding 100 mu L of the protein coating into each well, and incubating the protein coating at the temperature of 4 ℃ overnight; taking duck serum as a primary antibody, taking HRP-conjugated goat anti-duck IgG (diluted 1:5000) as a secondary antibody, measuring the value of a sample OD450 by using an enzyme-labeled instrument after color development, and obtaining the antibody titer in serum by a standard curve. Cytokine levels in serum were detected using commercial IL-4, IL-10, IFN-gamma and IgG ELISA kits; immunoglobulin content in duck intestinal mucosal fluid was detected using a commercial sIgAELISA kit, as shown in FIG. 6.
Data were analyzed by two-way anova on multiple comparisons in GraphPad Prism 9.1. The experimental and control groups were compared (P < 0.05). In general, the concentrations of IgG, sIgA, IL-4 and IFN-gamma in the ducks fed MG1363/1+3VP1 recombinant lactococcus lactis were higher than those in the control group.
In the application, a lactobacillus constitutive expression vector is selected, a section of unique current identifiable secretion protein Usp45 signal peptide and anchoring motif pgsA' of lactobacillus is added in front of target protein VP1, the signal peptide has been proved to realize secretion expression in almost all lactococcus lactis, so that recombinant proteins can be attached to a membrane layer, excessive accumulation and degradation of heterologous proteins in cells are avoided, the heterologous proteins are secreted outside the cells in time, and meanwhile, the interference of carrier microorganism self proteins on the heterologous proteins is reduced. The pgsA' protein can anchor and express the exogenous protein on the bacterial cell wall, so that the expression rate of the exogenous protein on the surface of the lactic acid bacteria is effectively improved, the organism is effectively induced to generate mucosal immunity, and the common defects of the traditional vaccine are avoided: the inactivated vaccine can not effectively induce the organism to generate cellular immunity and mucous membrane immunity, and the attenuated vaccine has strong virulence. The recombinant lactococcus lactis capable of realizing extracellular secretion and expression is finally constructed, and the recombinant lactococcus lactis is prepared into freeze-dried powder for oral immunization, so that organisms can be promoted to produce protective antibodies earlier, and the death rate of duck groups is reduced.
The foregoing is a further detailed description of the invention in connection with certain preferred embodiments, so that those skilled in the art will readily understand and practice the invention and will not be deemed to be limited to such description. It will be apparent to those skilled in the art that several simple deductions or substitutions may be made without departing from the inventive concept. Accordingly, it is intended that all such modifications as would be within the scope of this invention be included within the scope of this invention.
Claims (10)
1. A recombinant lactococcus lactis strain, characterized by the deposit number: CGMCC No.26965.
2. A method for constructing a recombinant lactococcus lactis strain, comprising the steps of:
step 1: extracting genome RNA of type 1 and type 3 duck hepatitis A viruses, respectively carrying out reverse transcription, taking the obtained cDNA as a template, designing primers to respectively amplify two pathogenic VP1 gene fragments, connecting to form VP1 (1, 3) through homologous recombination, and amplifying by a specific primer to ensure that two ends of the sequence respectively contain pgsA' and eGPF gene homology arms;
step 2: extracting bacillus subtilis genome DNA, designing a primer for amplification to obtain pgsA' fragments with both ends respectively containing Usp45 and VP1 (1, 3) gene homology arms;
step 3: designing a primer to amplify by taking a T7g10L-Usp45-VP1-eGFP-pMG36e plasmid as a template to obtain a T7g10L-Usp45 fragment with both ends containing a pMG36e vector and pgsA' homology arms;
step 4: designing primers to amplify eGFP fragments containing VP1 genes and pMG36e homology arms at two ends by taking pEGFP-C3 plasmid as a template;
step 5: connecting the gene fragments obtained in the steps 1, 2, 3 and 4 through homologous recombination to obtain a fusion fragment T7g10L-Usp45-pgsA' -1VP1-3VP1-eGFP;
step 6: the fusion fragment and a linearization vector pMG36e are connected through homologous recombination to obtain a recombinant plasmid Usp45-pgsA' -1VP1-3VP1-eGFP-pMG36e;
step 7: transforming the recombinant plasmid obtained in the step 6 into escherichia coli DH5 alpha, screening positive transformants by erythromycin resistance, picking single colony for PCR verification and sequencing, and culturing the correct recombinant single colony at 37 ℃ to obtain a cloning plasmid;
step 8: and (3) electrically transferring the recombinant plasmid obtained in the step (7) into lactococcus lactis MG1363, screening positive strains by using erythromycin resistance, selecting single colony PCR for verification, preserving the correct strains, and storing the strains at-80 ℃.
3. The method of claim 2, wherein in step 1, the DHAV-1/VP1 fragment is amplified using a primer 3F/3R pair, and the sequence of primer 3F/3R is as follows: 5'-GAAAATTTCGTACCAGAAAGTCGGTGATTCTAACCAGTTAGG-3';
5'-CCAAGCTGATTAGAATCACCTTCAATTTCCAAATTGAGTT-3'；
the DHAV-3/VP1 fragment was amplified using primer 4F/4R, the sequence of primer 4F/4R was as follows:
5'-CTCAATTTGGAAATTGAAGGTGATTCTAATCAGCTTGGTGAT-3'；
5'-CAGCTCCTCGCCCTTGCTCACTTCAATTTCTAGATGGAGCT-3'；
VP1 (1, 3) was then amplified specifically using two pairs of 3F/3R and 4F/4R primers, so that the sequence contained homologous arms of the pgsA' and eGPF genes at both ends, respectively.
4. The method of constructing a recombinant lactococcus lactis strain according to claim 2, wherein the primer 2F/2R is used to amplify the fragment pgsA' in step 2, and the primer 2F/2R has the following sequence:
5'-CGTTGTCAGGTGTTTACGCTAAAAAAGAACTGAGCTTTC-3'；
5'-CCTAACTGGTTAGAATCACCGACTTTCTGGTACGAAATTTTC-3'。
5. the method of claim 4, wherein the primer 5F/5R is used to amplify the fragment eGFP in step 4, and the primer 1F/1R has the following sequence:
5'-GAGCTCCATCTAGAAATTGAAGTGAGCAAGGGCGAGGAGCTG-3'；
5'-TTCAGACTTTGCAAGCTTTCAATGGTGATGGTGATGATGGTTA-3'。
6. the method of constructing a recombinant lactococcus lactis strain according to claim 5, wherein the fusion fragment obtained in step 5 has a DNA sequence shown in SEQ No. 1.
7. An immune microecological preparation for duck viral hepatitis, comprising the recombinant lactococcus lactis according to any one of claims 1-7.
8. The immune microecological preparation for duck viral hepatitis according to claim 8, wherein the concentration of recombinant lactococcus lactis bacteria in the immune microecological preparation is as follows: 5.0X10 8 cfu/mL。
9. The immune microecological formulation of claim 8, wherein the immune microecological formulation is a vaccine for administration by oral gavage.
10. The preparation method of the duck viral hepatitis immune microecological preparation is characterized by comprising the following steps of: respectively amplifying VP1 genes from the duck hepatitis A virus genome type 1 and the duck hepatitis A virus genome type 3, and simultaneously amplifying a signal peptide Usp45 sequence and a pgsA' sequence; then, the DHAV-1VP1 and DHAV-3VP1 genes are inserted into a pMG36e vector in series through a homologous recombination method, an anchor sequence pgsA' is introduced, a recombinant plasmid vector carrying DHAV-1VP1 and DHAV-3VP1 fusion genes is constructed, the plasmid vector is introduced into lactococcus lactis MG1363, recombinant lactococcus lactis is constructed, and the recombinant lactococcus lactis containing the DHAV-VP1 (1, 3) fusion genes is utilized to prepare the duck viral hepatitis immune microecologics.
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