CN118812728A - A preparation method and application of recombinant Lactococcus lactis strain - Google Patents

Abstract

The invention discloses a preparation method and application of a recombinant lactococcus lactis strain, and belongs to the technical field of genetic engineering. The invention constructs the lactococcus lactis strain for expressing the novel goose parvovirus fusion protein, and the strain can efficiently fix the fusion protein on the surface of the recombinant lactococcus lactis and can stably express without induction. According to the invention, the VP2-123 is constructed by utilizing the antigen epitopes (VP 2-1, VP2-2 and VP 2-3) of VP2 in series, the immunogenicity of VP2-123 is similar to that of VP2 with full-length sequence, and the virus proliferation of NGPV is effectively prevented by constructing recombinant bacteria by using the antigen epitopes, the length of VP2-123 is reduced, the gene replication efficiency is improved, the error probability in the protein expression process is reduced, and the use amount and cost of raw materials in the production process are reduced. The recombinant lactococcus lactis microecological preparation is used for oral immunization, so that the organism is promoted to generate protective antibodies, and the death rate of duck groups is reduced.

Description

A preparation method and application of recombinant Lactococcus lactis strain

Technical Field

The present invention relates to the technical field of genetic engineering, and in particular to a preparation method of a recombinant Lactococcus lactis strain and application thereof.

Background Art

Duck-derived novel goose parvovirus (NGPV) is mainly caused by infection with novel goose parvovirus, and is one of the most important virulent infectious diseases that endanger the duck farming industry. The disease is characterized by acute liver damage, and the sick duckling rate of ducklings is as high as 80%. "CN 108939063 B A triple inactivated vaccine for Muscovy ducks" provides a triple inactivated vaccine for duck type 2 adenovirus, goose parvovirus disease, and Muscovy duck parvovirus disease, providing immune protection for ducks. "CN 103272230 B A triple live vaccine for Muscovy ducks" discloses a triple vaccine for Muscovy ducks. The triple vaccine for Muscovy ducks uses the attenuated MWCA strain of Muscovy duck liver white spot disease, the attenuated P1 strain of Muscovy duck parvovirus, and the attenuated D strain of Muscovy duck-derived goose parvovirus as seed viruses. Immunization can prevent and control Muscovy duck gosling plague. Although vaccines and egg yolk antibodies are available for the prevention and treatment of the disease, immune failure occurs from time to time, which seriously hinders the healthy development of my country's duck farming industry. Current commercial vaccines have defects in disease prevention and control, such as pathogen mutation and failure of immunity caused by attenuated live vaccines.

Probioties, also called Bigone or probiotics, refer to preparations made by special processes using the principles of microecology, using probiotics or probiotic growth-promoting substances that are beneficial and harmless to the host. Probioties can be used for immunization through drinking water feeding, which is considered to be an acceptable immunization method. The administration route of probioties is convenient and safe, and large-scale immunization can be carried out quickly at the same time, which can avoid stress on the body, save labor, reduce immunization costs, and protect the body against pathogen invasion. Although probioties have the above advantages, there are currently no reports on the use of new goose parvovirus to prepare Lactococcus lactis probioties for virus prevention and control.

Summary of the invention

In view of the above prior art, the object of the present invention is to provide a preparation method of recombinant Lactococcus lactis and its application.

To achieve the above object, the present invention adopts the following technical solutions:

In a first aspect of the present invention, a NGPV antigen is provided, wherein the NGPV antigen contains the VP2-123 gene, and the nucleotide sequence of the VP2-123 gene is shown in SEQ ID NO:4.

The second aspect of the present invention provides a recombinant plasmid containing the NGPV antigen gene.

The third aspect of the present invention provides a recombinant Lactococcus lactis strain, wherein the recombinant Lactococcus lactis strain contains the NGPV antigen gene.

The fourth aspect of the present invention provides a method for preparing the recombinant Lactococcus lactis strain, comprising the following steps:

T7g10L-Usp45-pgsA', the target gene and eGFP were fused together to obtain a fusion gene with pMG36e homology arms; the fusion gene was inserted into the plasmid pMG36e to obtain a recombinant plasmid; the recombinant plasmid was transformed into Escherichia coli competent cells; positive plasmids were screened and secondary transformed into Lactococcus lactis MG1363 to obtain a recombinant Lactococcus lactis strain;

The target gene is the VP2-123 gene, and the nucleotide sequence of the VP2-123 gene is shown in SEQ ID NO:4.

The fifth aspect of the present invention provides the use of the recombinant antigen, the recombinant plasmid or the recombinant Lactococcus lactis strain in the preparation of a recombinant Lactococcus lactis probiotic preparation.

Furthermore, the recombinant Lactococcus lactis proecological preparation can be used to prepare a product for treating or preventing a novel goose parvovirus.

The sixth aspect of the present invention provides a microecological preparation containing the recombinant antigen, the recombinant plasmid or the recombinant Lactococcus lactis strain.

The seventh aspect of the present invention provides the use of the probiotic preparation of the recombinant Lactococcus lactis strain in the preparation of a product for treating or preventing a novel goose parvovirus.

In an eighth aspect of the present invention, a method for preparing the probiotic preparation is provided, wherein the recombinant Lactococcus lactis strain is freeze-dried to obtain the probiotic preparation.

Beneficial effects of the present invention:

The present invention successfully constructed a Lactococcus lactis strain expressing a novel goose parvovirus VP2 (VP2-1, VP2-2, VP2-3, VP2-123) fusion protein, which can efficiently fix the fusion protein on the surface of the recombinant Lactococcus lactis and can stably express it without induction. After oral immunization, significantly increased specific antibody IgG and intestinal mucosal sIgA were detected in the recombinant lactic acid bacteria strain duck, which significantly reduced the incidence and stiff duck rate of ducks infected with NGPV. After oral immunization, duck blood routine showed that the white blood cells and lymphocytes of the recombinant lactic acid bacteria strain duck group were upregulated for a period of time compared with other groups, indicating that the bacteria enhanced the immune response of the body's immune system; the viral load in the liver, spleen, kidney, lung, intestine and blood of the ducklings decreased, and the pathological section showed that the degree of tissue and organ damage in the ducklings of the recombinant bacteria group was significantly weakened, indicating that the recombinant bacteria effectively prevented the viral proliferation of NGPV.

In addition, the inventors constructed VP2-123 using the antigenic epitopes of VP2 (VP2-1, VP2-2, VP2-3) in series. Although the best immune condition was still the MG1363/VP2 group, the MG1363/VP2-123 group was not much different from it. Since VP2-123 only used three antigenic epitopes in the VP2 gene, the immunogenicity of VP2-123 was close to that of using the full-length sequence VP2, but the length of VP2-123 was reduced, so the replication efficiency of the shorter gene was improved, the error probability in the protein expression process was reduced, and the amount and cost of raw materials used in the production process were reduced.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a diagram showing the construction of a recombinant plasmid in E. coli DH5α.

Figure 2 shows the results of fluorescence microscopy, where A is the bright field of recombinant Lactococcus lactis MG1363/VP2; B is the fluorescence field of recombinant Lactococcus lactis MG1363/VP2; C is the bright field of recombinant Lactococcus lactis MG1363/VP2-123; D is the fluorescence field of recombinant Lactococcus lactis MG1363/VP2-123; E is the bright field of Lactococcus lactis containing pMG36e; F is the fluorescence field of Lactococcus lactis pMG36e. Scale bar: 5 μm.

Figure 3 is the Western blot analysis result of induced expression of NGPV-VP2, where M: protein molecular weight standard; 1: Lactococcus lactis MG1363/pMG36e; 2: recombinant Lactococcus lactis MG1363/VP2; 3: Lactococcus lactis MG1363.

Figure 4 is a graph showing the Dot Blot analysis results of recombinant Lactococcus lactis MG1363/VP2, MG1363/VP2-1, MG1363/VP2-2, MG1363/VP2-3 and MG1363/VP2-123.

Figure 5 shows the immune levels in the serum or intestinal samples of ducklings; the changes in the concentration levels of IL-2, IL-4, IFN-γ, IL-10, IgG and sIgA, *p<0.05; **p<0.01; ***p<0.001.

Figure 6 shows the weight and beak length of the ducklings during their growth.

Figure 7 shows the weight of various tissues and organs of ducklings, including liver, spleen, kidney, lung, and intestine tissues, *p<0.05; **p<0.01; ***p<0.001.

Fig. 8 Virus load in duckling tissues, including liver, spleen, kidney, lung, intestinal tissue, and blood, *p<0.05; **p<0.01; ***p<0.001.

Figure 9 is a blood routine test chart. WBC stands for white blood cells, RBC stands for red blood cells, LYM stands for lymphocytes, *p<0.05; **p<0.01; ***p<0.001.

Figure 10 shows the pathological histological changes of the liver. Groups A1 and A2 are control groups, groups B1 and B2 are MG1363/VP2 groups, groups C1 and C2 are MG1363/VP2-123 groups, and groups D1 and D2 are MG1363/MG36e groups. A1, B1, C1, and D1 are before the challenge, 7 days old. A2, B2, C2, and D2 are 8 days after the challenge, 16 days old. Scale bar: 5 μm.

Figure 11 shows the pathological histological changes of spleen. Groups A1 and A2 are control groups, groups B1 and B2 are MG1363/VP2 groups, groups C1 and C2 are MG1363/VP2-123 groups, and groups D1 and D2 are MG1363/MG36e groups. A1, B1, C1, and D1 are before the challenge, 7 days old. A2, B2, C2, and D2 are 8 days after the challenge, 16 days old. Scale bar: 5 μm.

Figure 12 shows the pathological histological changes of the kidney. Groups A1 and A2 are control groups, groups B1 and B2 are MG1363/VP2 groups, groups C1 and C2 are MG1363/VP2-123 groups, and groups D1 and D2 are MG1363/MG36e groups. A1, B1, C1, and D1 are before the challenge, 7 days old. A2, B2, C2, and D2 are 8 days after the challenge, 16 days old. Scale bar: 5 μm.

Figure 13 shows the pathological histological changes in the lungs. Groups A1 and A2 are control groups, groups B1 and B2 are MG1363/VP2 groups, groups C1 and C2 are MG1363/VP2-123 groups, and groups D1 and D2 are MG1363/MG36e groups. A1, B1, C1, and D1 are before the challenge, 7 days old. A2, B2, C2, and D2 are 8 days after the challenge, 16 days old. Scale bar: 5 μm.

DETAILED DESCRIPTION

It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

The experimental materials used in the examples of the present invention without specific description are all conventional experimental materials in the field and can be purchased through commercial channels. The source information of the enhancer T7g10L, amplification signal peptide Usp45 sequence, pMG36e plasmid, and Lactococcus lactis MG1363 used can be found in the previous paper of this research group "Construction of recombinant Lactococcus lactis strain expressing new duck reovirus σC protein and evaluation of immune effect" (Zhang Hongrui, Shandong Agricultural University).

In the present application, the NGPV (VP2, VP2-1, VP2-2, VP2-3, VP2-123) gene is inserted into the pMG36e vector by homologous recombination, and the anchor sequence pgsA' is introduced to construct a recombinant plasmid vector carrying the NGPV (VP2, VP2-1, VP2-2, VP2-3, VP2-123) fusion gene, and the plasmid vector is introduced into Lactococcus lactis MG1363 to construct a recombinant Lactococcus lactis. The recombinant Lactococcus lactis containing the NGPV (VP2, VP2-1, VP2-2, VP2-3, VP2-123) fusion gene is used to prepare a new duck-derived goose parvovirus immune microecological preparation.

Example 1: Construction of a recombinant plasmid carrying the NGPV gene

The VP2 gene (GenBank: MW811196.1), VP2-1, VP2-2, VP2-3, and VP2-123 genes were amplified from NGPV-positive diseased materials preserved in the laboratory, and the signal peptide Usp45 sequence and pgsA' sequence were amplified at the same time. The pMG36e plasmid was used as a vector plasmid to construct a recombinant plasmid carrying the NGPV (VP2, VP2-1, VP2-2, VP2-3, VP2-123) fusion gene.

VP2-1 gene nucleotide sequence (SEQ ID NO: 1):

ACGGCTCCTGCAAAAAAAAATACAGGGAAGCTTACTGACCATTACCCAGTAGTTAA

GAAGCCTAAACTCACCGAGGAAGTCAGTGCGGGAGGTGGTAGCAGTGTCGTACAA

GACGGAGGAGCCACCGCGGAGGGCACCGAACCTGTGGCAGCATCTGAAATGGCAG

AGGGAGGAGGCGGAGCTATGGGCGACTCTTCAGGGGGTGCCGATGGAGTGGGTAA

TGCCTCGGGAAATTGGCATTGCGATTCCCAATGGATGGGAAACACAGTCATCACAA

AGACCACCAGAACCTGGGTCCTGCCAAGCTACAACAATCACATCTACAAAGCAATT

ACCAGTGGAACCTCTCAAGATGCAAATGTCCAGTATGCTGGATACAGTACCCCCTG

GGGGTACTTTGATTTCAATCGCTTCCACTGCCACTTCTCCCCTAGAGACTGGCAGAG

ACTTATCAACAACCACTGGGGAATCAGGCCCAAGTCTCTTAAATTCAAGATCTTCA

ATGTTCAAGTCAAGGAAGTCACAACGCAGGATCAGACAAAGACCATTGCAAACAA

TCTCACCTCAACAATCCAAGTTTTTACGGATGATGAGCACCAACTCCCGTATGTCCT

GGGCTCGGCTACGGAAGGGACCATGCCGCCGTTCCCGTCGGATGTATATGCCCTGC

CGCAGTACGGGTACTGCACAATGCACACCAACCAGAATGGAGCACGGTTCAATGAC

CGTAGCGCATTCTACTGCTTAGAGTACTTCCCTAGTCAGATGCTGAGAACAGGTAACAACTTTGAGTTCACATTTGACTTTGAA.

VP2-2 gene nucleotide sequence (SEQ ID NO: 2):

TCTCTTAAATTCAAGATCTTCAATGTTCAAGTCAAGGAAGTCACAACGCAGGATCA

GACAAAGACCATTGCAAACAATCTCACCTCAACAATCCAAGTTTTTACGGATGATG

AGCACCAACTCCCGTATGTCCTGGGCTCGGCTACGGAAGGGACCATGCCGCCGTTC

CCGTCGGATGTATATGCCCTGCCGCAGTACGGGTACTGCACAATGCACACCAACCA

GAATGGAGCACGGTTCAATGACCGTAGCGCATTCTACTGCTTAGAGTACTTCCCTAG

TCAGATGCTGAGAACAGGTAACAACTTTGAGTTCACATTTGACTTTGAAGAAGTTCC

TTTCCACAGCATGTTCGCTCATTCACAGGACTTAGACAGGCTTATGAACCCCCTAGT

GGATCAATACCTCTGGAATTTCAATGAGGTAGACAGCAACAGAAATGCTCAATTTA

AAAAAGCTGTGAAAGGGGCTTATGGCACCATGGGCCGCAATTGGCTGCCGGGACCT

AAATTCCTGGATCAGAGAGTTAGGGCCTACCCAGGAGGAACAGACAATTATGCAAA

CTGGAACATCTGGAATAATGGGAACAAGGTGAATTTAAAGGACAGGCAGTATCTCC

TACAACCCGGACCTGTGTCAGCTACTCACACAGAAGGGGAGGCTTCCAGCATCCCA

GCTCAGAATATTTTAGGGATAGCTAAAGATCCATACAGATCTGGCAGCACTACAGC

AGGAATAAGTGATATTATGGTCACGGACGAGCAGGAAGTAGCACCCACGAATGGA

GTAGGGTGGAAACCATATGGTAGGACTGTAACGAATGAACAAAACACTACTACAG

CTCCTACAAGTTCAGATCTGGATGTTCTTGGAGCTTTACCAGGAATGGTGTGGCAGAAC.

VP2-3 gene nucleotide sequence (SEQ ID NO: 3):

GCCGATGGAGTGGGTAATGCCTCGGGAAATTGGCATTGCGATTCCCAATGGATGGG

AAACACAGTCATCACAAAGACCACCAGAACCTGGGTCCTGCCAAGCTACAACAATC

ACATCTACAAAGCAATTACCAGTGGAACCTCTCAAGATGCAAATGTCCAGTATGCT

GGATACAGTACCCCCTGGGGGTACTTTGATTTCAATCGCTTCCACTGCCACTTCTCC

CCTAGAGACTGGCAGAGACTTATCAACAACCACTGGGGAATCAGGCCCAAGTCTCT

TAAATTCAAGATCTTCAATGTTCAAGTCAAGGAAGTCACAACGCAGGATCAGACAA

AGACCATTGCAAACAATCTCACCTCAACAATCCAAGTTTTTACGGATGATGAGCAC

CAACTCCCGTATGTCCTGGGCTCGGCTACGGAAGGGACCATGCCGCCGTTCCCGTC

GGATGTATATGCCCTGCCGCAGTACGGGTACTGCACAATGCACACCAACCAGAATG

GAGCACGGTTCAATGACCGTAGCGCATTCTACTGCTTAGAGTACTTCCCTAGTCAGA

TGCTGAGAACAGGTAACAACTTTGAGTTCACATTTGACTTTGAAGAAGTTCCTTTCC

ACAGCATGTTCGCTCATTCACAGGACTTAGACAGGCTTATGAACCCCCTAGTGGATC

AATACCTCTGGAATTTCAATGAGGTAGACAGCAACAGAAATGCTCAATTTAAAAAA

GCTGTGAAAGGGGCTTATGGCACCATGGGCCGCAATTGGCTGCCGGGACCTAAATT

CCTGGATCAGAGAGTTAGGGCCTACCCAGGAGGAACAGACAATTATGCAAACTGG

AACATCTGGAATAATGGGAACAAGGTGAATTTAAAGGACAGGCAGTATTCCCTACAA.

VP2-123 gene nucleotide sequence (SEQ ID NO: 4):

ACGGCTCCTGCAAAAAAAAATACAGGGAAGCTTACTGACCATTACCCAGTAGTTAA

GAAGCCTAAACTCACCGAGGAAGTCAGTGCGGGAGGTGGTAGCAGTGTCGTACAA

GACGGAGGAGCCACCGCGGAGGGCACCGAACCTGTGGCAGCATCTGAAATGGCAG

AGGGAGGAGGCGGAGCTATGGGCGACTCTTCAGGGGGTGCCGATGGAGTGGGTAA

TGCCTCGGGAAATTGGCATTGCGATTCCCAATGGATGGGAAACACAGTCATCACAA

AGACCACCAGAACCTGGGTCCTGCCAAGCTACAACAATCACATCTACAAAGCAATT

ACCAGTGGAACCTCTCAAGATGCAAATGTCCAGTATGCTGGATACAGTACCCCCTG

GGGGTACTTTGATTTCAATCGCTTCCACTGCCACTTCTCCCCTAGAGACTGGCAGAG

ACTTATCAACAACCACTGGGGAATCAGGCCCAAGTCTCTTAAATTCAAGATCTTCA

ATGTTCAAGTCAAGGAAGTCACAACGCAGGATCAGACAAAGACCATTGCAAACAA

TCTCACCTCAACAATCCAAGTTTTTACGGATGATGAGCACCAACTCCCGTATGTCCT

GGGCTCGGCTACGGAAGGGACCATGCCGCCGTTCCCGTCGGATGTATATGCCCTGC

CGCAGTACGGGTACTGCACAATGCACACCAACCAGAATGGAGCACGGTTCAATGAC

CGTAGCGCATTCTACTGCTTAGAGTACTTCCCTAGTCAGATGCTGAGAACAGGTAAC

AACTTTGAGTTCACATTTGACTTTGAAGAAGTTCCTTTCCACAGCATGTTCGCTCATT

CACAGGACTTAGACAGGCTTATGAACCCCCTAGTGGATCAATACCTCTGGAATTTC

AATGAGGTAGACAGCAACAGAAATGCTCAATTTAAAAAAGCTGTGAAAGGGGCTT

ATGGCACCATGGGCCGCAATTGGCTGCCGGGACCTAAATTCCTGGATCAGAGAGTT

AGGGCCTACCCAGGAGGAACAGACAATTATGCAAACTGGAACATCTGGAATAATG

GGAACAAGGTGAATTTAAAGGACAGGCAGTATTCCCTACAACCCGGACCTGTGTCA

GCTACTCACACAGAAGGGGAGGCTTCCAGCATCCCAGCTCAGAATATTTTAGGGAT

AGCTAAAGATCCATACAGATCTGGCAGCACTACAGCAGGAATAAGTGATATTATGG

TCACGGACGAGCAGGAAGTAGCACCCACGAATGGAGTAGGGTGGAAACCATATGG

TAGGACTGTAACGAATGAACAAAACACTACTACAGTCCCTACAAGTTCAGATCTGG

ATGTTCTTGGAGCTTTACCAGGAATGGTGTGGCAGAACAGAGATATATATCTGCAG

GGACCTATTTGGGCAAAAATACCGAAGACTGATGGCAAATTCCATCCTTCTCCAAA

TCTCGGAGGATTTGGCCTGCACAATCCACCACCACAGGTCTTCATCAAGAATACAC

CAGTACCTGCAGACCCTCCAGTAGAATATGTGAACCAGAAGTGGAACTCCTACATA

ACTCAATACTCTACAGGCCAGTGTACAGTAGAAATGGTGTGGGAGCTGAGAAAAGAGAATTCAAAGAGATGGAACCCAGAAATC.

pgsA' gene nucleotide sequence (SEQ ID NO:5):

ATGAAAAAAGAACTGAGCTTTCATGAAAAGCTGCTAAAGCTGACAAAACAGCAAA

AAAAGAAAACCAATAAGCACGTATTTATTGCCATTCCGATCGTTTTTGTCCTTATGT

TCGCTTTCATGTGGGCGGGAAAAGCGGAAACGCCGAAGGTCAAAACGTATTCTGAC

GACGTACTCTCAGCCTCATTTGTAGGCGATATTATGATGGGACGCTATGTTGAAAAA

GTAACGGAGCAAAAAGGGGCAGACAGTATTTTTCAATATGTTGAACCGATCTTTAG

AGCCTCGGATTATGTAGCAGGAAACTTTGAAAACCCGGTAACCTATCAAAAGAATT

ATAAACAAGCAGATAAAGAGATTCATCTGCAGACGAATAAGGAATCAGTGAAAGT

CTTGAAGGATATGAATTTCACGGTTCTCAACAGCGCAAACAACCACGCAATGGATT

ACGGCGTTCAGGGCATGAAAGATACGCTTGGAGAATTTGCGAAGCAAAACCTTGAT

ATCGTTGGAGCGGGATACAGCTTAAGTGATGCGAAAAAGAAAATTTCGTACCAGAA

AGTCAACGGGGTAACGATTGCGACGCTTGGCTTTACCGATGTGTCCGGGAAAGGTT

TCGCGGCTAAAAAGAATACGCCGGGCGTGCTGCCCGCAGATCCTGAAATCTTCATC

CCTATGATTTCAGAAGCGAAAAAACATGCTGACATTGTTGTTGTGCAGTCACACTG

GGGCCAAGAGTATGACAATGATCCAAACGACCGCCAGCGCCAGCTTGCAAGAGCC

ATGTCTGATGCGGGAGCTGACATCATCGTCGGCCATCATCCGCACGTCTTAGAACC

GATTGAAGTATATAACGGAACCGTCATTTTCTACAGCCTCGGCAACTTTGTCTTTGA

CCAAGGCTGGACGAGAACAAGAGACAGTGCACTGGTTCAGTATCACCTGAAGAAA

AATGGAACAGGCCGCTTTGAAGTGACACCGATCGATATCCATGAAGCGACACCTGC

ACCTGTGAAAAAAGACAGCCTTAAACAGAAAACCATTATTCGCGAACTGACGAAA

GACTCTAATTTCGCTTGGAAAGTAGAAGACGGAAAACTGACGTTTGATATTGATCATAGTGACAAACTAAAATCTAAATAA.

The PCR primers for amplifying T7g10L-Usp45-pgsA' are 1F (nucleotide sequence SEQ ID NO: 6) and 1R (nucleotide sequence SEQ ID NO: 7), the PCR primers for VP2 gene are 2F (nucleotide sequence SEQ ID NO: 8) and 2R (nucleotide sequence SEQ ID NO: 9), the PCR primers for eGFP gene are 3F (nucleotide sequence SEQ ID NO: 10) and 3R (nucleotide sequence SEQ ID NO: 11), and the PCR primers for pMG36e are 4F (nucleotide sequence SEQ ID NO: 12) and 4R (nucleotide sequence SEQ ID NO: 13). The specific sequences are shown in Table 1 below, wherein an eGFP tag is added to the end of the target gene primer, and the universal primer of pMG36e is selected as the identification primer.

Table 1 PCR primer sequences

Using plasmid T7g10L-Usp45-pgsA'-σC-eGFP-pMG36e as a template, primers 1F/1R were used to amplify the T7g10L-Usp45-pgsA' fragment; using plasmid 18-T-VP2 as a template, primers 2F/2R were used to amplify the NGPV-VP2 fragment; using plasmid T7g10L-Usp45-pgsA'-σC-eGFP-pMG36e as a template, primers 3F/3R were used to amplify the fragment eGFP.

T7g10L-Usp45-pgsA', VP2, and eGFP were fused together to obtain the fusion gene T7g10L-Usp45-pgsA'-VP2-eGFP with pMG36e homology arms. The fusion process is shown in Figure 1.

The plasmid and fusion gene were double-digested with restriction endonucleases Xba I and Hind III, and the final fragment was inserted into plasmid pMG36e. The recombinant plasmid was transformed into E. coli DH5α competent cells.

Prepare competent cells of Lactococcus lactis MG1363 in advance. Transfer the plasmid T7g10L-Usp45-pgsA'-VP2-eGFP-pMG36e into competent cells by electroporation technology, and culture the cells at a concentration of 1.0 μg/mL in GM17 agar medium containing erythromycin. The screened positive plasmid is secondary transformed into Lactococcus lactis MG1363. The positive recombinant MG1363 strain contains T7g10L-Usp45-pgsA'-VP2-eGFP-pMG36e and is renamed MG1363/VP2. Lactococcus lactis MG1363/pMG36e is used as a negative control. The recombinant Lactococcus lactis was observed under a Nikon upright fluorescence microscope, as shown in Figure 2.

At the same time, the VP2 gene was replaced with the antigenic epitope 1 fragment (VP2-1), the antigenic epitope 2 fragment (VP2-2), the antigenic epitope 3 fragment (VP2-3) and the tandem antigenic epitope fragment (VP2-123) of VP2-1, VP2-2 and VP2-3 to construct a recombinant plasmid, the method was the same as above, and the primer sequences were shown in Table 2. Among them, 1F, 1R, 2F, and 4R are the same as Table 1. The primers for VP2-1 are 2R (nucleotide sequence SEQ ID NO: 14), 3F (nucleotide sequence SEQ ID NO: 15), 3R (nucleotide sequence SEQ ID NO: 16), and 4F (nucleotide sequence SEQ ID NO: 17). The primers for VP2-2 are 2R (nucleotide sequence SEQ ID NO: 18), 3F (nucleotide sequence SEQ ID NO: 19), 3R (nucleotide sequence SEQ ID NO: 20), and 4F (nucleotide sequence SEQ ID NO: 21). The primers for VP2-3 are 2R (nucleotide sequence SEQ ID NO: 22), 3F (nucleotide sequence SEQ ID NO: 23), 3R (nucleotide sequence SEQ ID NO: 24), and 4F (nucleotide sequence SEQ ID NO: 25). The primers for VP2-123 are 2R (nucleotide sequence SEQ ID NO: 26), 3F (nucleotide sequence SEQ ID NO: 27), 3R (nucleotide sequence SEQ ID NO: 28), and 4F (nucleotide sequence SEQ ID NO: 29).

The recombinant Lactococcus lactis were named MG1363/VP2-1, MG1363/VP2-2, MG1363/VP2-3 and MG1363/VP2-123.

Table 2 PCR primer sequences for amplifying VP2-related genes

Example 2: Identification of recombinant Lactococcus lactis fusion gene expression

First, recombinant Lactococcus lactis MG1363/VP2, MG1363/VP2-1, MG1363/VP2-2, MG1363/VP2-3, MG1363/VP2-123 and MG1363/pMG36e were added to GM17 medium without erythromycin and cultured at 37°C for 18 hours. The bacterial solution was centrifuged at 5000rpm for 10 minutes to separate the bacterial precipitate and supernatant. The bacterial precipitate was resuspended with TES solution, allowed to stand at 37°C for 30 minutes, centrifuged again, the supernatant was discarded and resuspended with TE buffer, centrifuged at 5000rpm for 10 minutes, and the precipitate was resuspended with ultrapure water and repeatedly frozen and thawed 5 times. Subsequently, it was centrifuged at 4°C and the precipitate was resuspended with PBS solution. Finally, the target protein was isolated using Anti-GFP magnetic beads. The isolated target protein was subjected to Western blotting to detect whether the target protein existed on the cell wall. Using eGFP-Tag mouse monoclonal antibody as primary antibody and HRP-conjugated goat anti-mouse IgG as secondary antibody, the theoretical molecular weight of the target fusion protein is 122 kDa, and the results of Western blot assay are shown in Figure 3. At the same time, Dot Blot analysis was performed on recombinant Lactococcus lactis MG1363/VP2, MG1363/VP2-1, MG1363/VP2-2, MG1363/VP2-3 and MG1363/VP2-123.

The results are shown in FIG4 , and all of the five groups of recombinant Lactococcus lactis fusion genes analyzed by Dot Blot were expressed.

The recombinant Lactococcus lactis MG1363/VP2, MG1363/VP2-1, MG1363/VP2-2, MG1363/VP2-3 and MG1363/VP2-123 were freeze-dried to prepare duck-derived new goose parvovirus immune microecological preparations for subsequent use.

Example 3: Immunoprotective effect of recombinant Lactococcus lactis

415 one-day-old Cherry Valley ducks were raised in a sterile isolation box, and the required food and water were sterilized by high pressure in advance. On the first day of the experiment, 9 healthy ducklings were randomly selected, and jugular vein blood and intestinal specimens were taken as references for oral immune indicators. The remaining ducklings were randomly divided into 9 groups (Group A-Group I): Group A (blank group, 61), Group B (MG1363/VP2 group, 45), Group C (MG1363/pMG36e group, 45), Group D (MG1363 group, 45), Group E (yolk antibody group, 30), Group F (MG1363/VP2-1 group, 45), Group G (MG1363/VP2-2 group, 45), Group H (MG1363/VP2-3 group, 45) and Group I (MG1363/VP2-123 group, 45). In order to reduce the stress of one-day-old ducklings during transportation, all groups of ducklings were fed only sterile water and sterile duck feed. When the ducklings were 2 to 8 days old, at the same time every day, the ducklings in groups B, C, D, F, G, H, and I were orally immunized with MG1363/VP2 recombinant lactic acid bacteria, MG1363/pMG36e empty lactic acid bacteria, MG1363 empty bacteria, MG1363/VP2-1 recombinant lactic acid bacteria, MG1363/VP2-2 recombinant lactic acid bacteria, MG1363/VP2-3 recombinant lactic acid bacteria, and MG1363/VP2-123 recombinant lactic acid bacteria, with a dose of 0.5 mL (7.3×10 ¹⁰ CFU/mL, taken from the freeze-dried and reconstituted microecological preparation in Example 3). Group A was used as a blank control group and was fed only sterile water and feed every day. For group E, in order to avoid the interference of maternal antibodies, each duck was intramuscularly injected with 0.5 mL of anti-NGPV yolk antibody at seven days of age. When the ducklings were eight days old, 10 healthy ducks were randomly selected from the blank group of group A, and each duck was orally infected with NGPV, 0.5 mL each time, to simulate the natural state of infection in other ducklings, and divided into groups A, B, C, D, E, F, G, H, and I, with 2 ducklings each. At this time, there were 32 ducklings in each group, and the infection of each group of ducklings was observed, and the weight, beak length and mortality of each group of ducklings were recorded in time. Venous blood and intestinal specimens were collected from ducklings at 4 days, 7 days, 10 days, 13 days, 16 days, and 19 days of age to detect blood routine, NGPV-specific antibodies in the blood, and viral load in the intestines. The liver, kidney, spleen, and lung were collected at 7 days, 10 days, 13 days, 16 days, and 19 days of age to detect NGPV-specific antibodies and various tissue lesions.

After immunization, 5 ducklings were randomly collected from each group at 2, 4, 7, 10, 13, 16 and 19 days of age to collect serum and duodenal samples. The duodenal samples were repeatedly rinsed with PBS buffer, the rinse was shaken on an oscillator for 1 hour, centrifuged at 12000rpm at 4℃ for 10 minutes, the supernatant was aspirated, and BSA was added to a final concentration of 0.1% to obtain the small intestinal mucosal fluid sample. The purified protein coating was diluted and added to a 96-well plate, 100 μL was added to each well, and incubated overnight at 4℃; duck serum was used as the primary antibody, and HRP-conjugated goat anti-duck IgG (1:5000 dilution) was used as the secondary antibody. After color development, the sample OD ₄₅₀ value was measured with an enzyme reader and the antibody titer in the serum was obtained through the standard curve. The levels of cytokines in serum were detected using commercial IL-2, IL-4, IL-10, IFN-γ, IgG and sIgA ELISA kits (Figure 5); the immunoglobulin content in the duck small intestinal mucosal fluid was detected using a commercial sIgA ELISA kit, as shown in Figure 7. A series of tests were continued on the experimental ducks, including the weight and beak length of the ducklings during growth (Figure 6), viral load detection of various tissues of the ducklings (Figure 8), blood routine test (Figure 9), and pathological histological changes of the liver (Figure 10), spleen (Figure 11), kidney (Figure 12), and lung (Figure 13).

Data were analyzed by two-way ANOVA for multiple comparisons in GraphPad Prism 9.1. The experimental group was compared with the control group (P<0.05). Overall, the concentrations of IgG, sIgA, IL-2, IL-4, and IFN-γ in ducks fed MG1363/VP2 recombinant Lactococcus lactis were higher than those in the control group and the tandem antigen epitope group. The weight of ducks fed MG1363/VP2 recombinant Lactococcus lactis increased steadily and the incidence of short beak was lower than that in other groups. The viral loads in organs such as liver, kidney, and lung in ducklings were lower than those in other groups. Blood routine showed that the ducklings in the MG1363/VP2 recombinant Lactococcus lactis group were the most stable. It is worth mentioning that in the immunization results expressing the NGPV full gene, tandem antigen epitope group 1, group 2, group 3 and tandem antigen epitope group 123, although the best immune situation is still the MG1363/VP2 group, the NGPV tandem antigen epitope 123 group (MG1363/VP2-123) is not much different from it. Since VP2-123 only uses three antigen epitopes in the VP2 gene, while the immunogenicity of VP2-123 is close to that of the full-length sequence VP2, the length of VP2-123 has been shortened, and the efficiency of the shorter gene has been improved during the replication process, which reduces the probability of errors in the protein expression process and reduces the amount and cost of raw materials used in the production process.

In this application, a lactic acid bacteria constitutive expression vector is selected, and a signal peptide of Usp45, the only secretory protein currently identifiable by lactic acid bacteria, and an anchoring motif pgsA' are added before the target protein (VP2, VP2-1, VP2-2, VP2-3, VP2-123). This signal peptide has been confirmed to achieve secretory expression in almost all lactococci, so that the recombinant protein can be attached to the membrane layer, avoiding excessive accumulation and degradation of heterologous proteins in the cell, and timely secreting the exogenous protein to the extracellular space, while also reducing the interference of the carrier microorganism's own protein on the heterologous protein. The pgsA' protein can anchor the exogenous protein to the bacterial cell wall, effectively increase the expression rate of the exogenous protein on the surface of lactic acid bacteria, effectively induce the body to produce mucosal immunity, and avoid the common shortcomings of traditional vaccines: inactivated vaccines cannot effectively induce the body to produce cellular immunity and mucosal immunity, and attenuated vaccines have the problem of virulence reversal. The present application constructs a recombinant Lactococcus lactis that can ultimately achieve extracellular secretory expression and prepares the recombinant Lactococcus lactis into a lyophilized powder for oral immunization, thereby prompting the body to produce protective antibodies earlier and reducing the mortality rate of duck flocks.

The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims (9)

1. The NGPV antigen is characterized in that the NGPV antigen contains a VP2-123 gene, and the nucleotide sequence of the VP2-123 gene is shown as SEQ ID NO. 4.

2. A recombinant plasmid comprising the NGPV antigen of claim 1.

3. A recombinant lactic acid bacterium strain, characterized in that, the recombinant lactococcus lactis strain contains the NGPV antigen according to claim 1.

4. A method for preparing a recombinant lactococcus lactis strain according to claim 3, comprising the steps of:

Fusion of T7g10L-Usp45-pgsA', target gene and eGFP together to obtain fusion gene with pMG36e homology arm; the fusion gene is inserted into a plasmid pMG36e to obtain a recombinant plasmid; transforming the recombinant plasmid into competent cells of E.coli; screening out positive plasmids and secondarily converting the positive plasmids into lactococcus lactis MG1363 to obtain a recombinant lactococcus lactis strain;

The target gene is VP2-123 gene, and the nucleotide sequence of the VP2-123 gene is shown as SEQ ID NO. 4.

5. Use of the recombinant antigen of claim 1, the recombinant plasmid of claim 2 or the recombinant lactococcus lactis strain of claim 3 in the preparation of a recombinant lactococcus lactis microecological preparation.

6. The use according to claim 5, characterized in that the recombinant lactococcus lactis microecological preparation can be used for the preparation of a new goose parvovirus product for the treatment or prevention.

7. A probiotic comprising the recombinant antigen of claim 1, the recombinant plasmid of claim 2 or the recombinant lactococcus lactis strain of claim 3.

8. Use of a probiotic of recombinant lactococcus lactis strains according to claim 7, for the preparation of a product for the treatment or prevention of novel goose parvovirus.

9. The method for preparing the microecological preparation according to claim 7, wherein the recombinant lactococcus lactis strain according to claim 3 is lyophilized to obtain the microecological preparation.