CN117535323A

CN117535323A - Recombinant lactobacillus vector for expressing coronavirus M protein epitope peptide, recombinant lactobacillus plantarum and application

Info

Publication number: CN117535323A
Application number: CN202311500872.XA
Authority: CN
Inventors: 王茂鹏; 邓安琦; 叶丹妮; 艾雪言; 邓玲聪; 唐秀兰; 陈文聪; 陈嘉豪; 刘宇航; 郭俊廷
Original assignee: Wenzhou University
Current assignee: Wenzhou University
Priority date: 2023-11-13
Filing date: 2023-11-13
Publication date: 2024-02-09

Abstract

The invention provides a fusion gene for expressing coronavirus M protein epitope peptide, a recombinant lactobacillus vector, recombinant lactobacillus plantarum and application thereof, and belongs to the technical field of molecular biology. A fusion gene expressing a coronavirus M protein epitope peptide, comprising a gene sequence encoding a signal peptide, a target gene, and an SCWA signal peptide. The invention also provides a recombinant lactobacillus vector containing the fusion gene and recombinant lactobacillus plantarum constructed by the recombinant lactobacillus plantarum vector, and results show that the recombinant lactobacillus plantarum has higher exogenous gene expression efficiency and good immunogenicity, has higher application value in the aspect of mucosal immunity, and lays a foundation for developing novel nasal drip vaccines of novel crown M protein peptide epitopes.

Description

Recombinant lactobacillus vector for expressing coronavirus M protein epitope peptide, recombinant lactobacillus plantarum and application

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a recombinant lactobacillus vector for expressing coronavirus M protein epitope peptide, recombinant lactobacillus plantarum and application thereof.

Background

Severe acute respiratory syndrome coronavirus 2 (Severe Acute Respiratory Syndrome Coronavirus, SARS-CoV-2), also known as "New coronavirus" due to genomic variation relative to previously discovered coronaviruses. SARS-CoV-2 is transmitted through respiratory droplets and aerosols, and most patients exhibit mild to moderate respiratory disease, with cough, fever, headache, myalgia and diarrhea. At present, no specific medicine for treating severe acute respiratory syndrome coronavirus 2 infection exists, and positive prevention by preparing related vaccines is a main measure for preventing and controlling coronavirus infection.

SARS-CoV and SARS-CoV-2 are members of the genus beta-coronavirus of the family Coronaviridae, having a single-stranded positive strand RNA genome of about 30kb encoding 16 nonstructural proteins, 9 helper proteins and 4 major viral structural proteins, namely spike protein (S), nucleocapsid protein (N), membrane protein (M) and envelope protein (E). Structural proteins are important targets for vaccine and antiviral drug development because they have an indispensable function of fusion and entry into host cells.

The epitope vaccine is prepared by using antigen epitopes, and depending on the prediction of T cell and B cell epitopes, the epitopes can trigger specific and protective immune responses, and is a vaccine development technology which is emerging in recent years, and is one of vaccine technologies with the most development prospect in the future, and the epitope vaccine has unique advantages in the prevention and treatment of diseases such as tumors, viruses and the like. Peptide epitope vaccines are now an emerging and effective antiviral tool, and several reports have taught that this method has various advantages such as effective prevention of ADE phenomenon, more economical and practical.

Most of the coronavirus peptide epitope vaccines developed at present are designed based on spike protein (S), however, more and more reports on SARS-CoV-2 variants have been recently made, and a variant virus of S antigen specific Neutralizing Antibody (NAB) resistant to SARS-CoV-2 has been developed. Thus, there is a need to develop a vaccine that allows for the possible emergence of SARS-CoV to induce an effective immune response against these NAB-resistant varieties. The membrane proteins (M proteins) of SARS-CoV and SARS-CoV-2 coronavirus are the most abundant structural proteins in coronaviruses, and are composed of 222 amino acids, which are highly conserved when used as antigens for developing SARS-CoV-2 vaccine. Immunization with full-length M protein has been reported to elicit neutralizing antibodies in SARS patients, better eliciting cellular immunity.

Lactic acid bacteria are a kind of bacillus-free, gram-positive bacteria (G) ⁺ ) And bacteria with lactic acid as fermentation products are collectively called, have the characteristics of probiotics, no endotoxin, adhesion, immune regulation and the like, and the characteristics of general safety (GRAS) make the bacteria widely applied in the fields of treating intestinal diseases, cancers, anti-infection, feed additives and the like. Therefore, the expression and delivery system dependent on the lactobacillus vector has the characteristics of safety, high efficiency and capability of inducing the mucosal immunity of the organism. The preparation of live bacteria vaccines by using lactobacillus as an expression host cell has been reported, however, the preparation of live bacteria vaccines is reduced in immunogenicity of epitope polypeptides due to the restriction of the secretion of antigen epitopes on the surface of lactobacillus.

Disclosure of Invention

Therefore, the invention aims to provide a fusion gene for expressing coronavirus M protein epitope peptide, and the composition of the fusion protein enables the expressed coronavirus M protein epitope peptide to have good surface display capability, so that the expression level of lactobacillus surface epitope polypeptide can be greatly improved, the antigen presenting efficiency is improved, and the immunogenicity of live bacteria vaccine is improved.

The invention provides a fusion gene for expressing coronavirus M protein epitope peptide, which comprises a secretion peptide sequence, a coding coronavirus M protein epitope peptide sequence and an SCWA signal peptide sequence.

Preferably, the encoding coronavirus M protein epitope peptide sequence comprises a coding gene sequence of MS2 phage capsid protein chimeric with coronavirus M protein epitope peptide;

the amino acid sequence of the coronavirus M protein epitope peptide is shown as SEQ ID NO. 2 and 7;

the signal peptide comprises 2145 secretion peptide or 3050 secretion peptide.

Preferably, the fusion gene further comprises a gene sequence encoding DCpep and HA tag proteins.

Preferably, the fusion gene includes a 2S fusion gene and a 3S fusion gene;

the 2S fusion gene is a gene sequence encoding the following fusion protein: 2145 secretion peptide-MS 2 phage capsid protein chimeric with coronavirus M protein epitope peptide-DCpep peptide-SCWA signal peptide-HA tag;

the 3S fusion gene is a gene sequence encoding the following fusion protein: 3050 secretion peptide-MS 2 phage capsid protein chimeric with coronavirus M protein epitope peptide-DCpep peptide-SCWA signal peptide-HA tag.

The invention provides a recombinant lactobacillus vector, which comprises the fusion gene.

The invention provides a recombinant lactobacillus plantarum which contains the fusion gene or the recombinant lactobacillus vector.

Preferably, the recombinant lactobacillus plantarum comprises an MS-2S strain and/or an MS-3S strain;

the MS-2S strain comprises the 2S fusion gene or a recombinant lactobacillus vector inserted with the 2S fusion gene;

the MS-3S strain comprises the 3S fusion gene or comprises a recombinant lactobacillus vector inserted with the 3S fusion gene.

The invention provides a method for culturing recombinant lactobacillus plantarum, which comprises the steps of inoculating the recombinant lactobacillus plantarum into an MRS liquid culture medium for culturing;

the culture temperature is 36-38 ℃;

the initial pH of the culture was 7.0.

Preferably, the MS-2S strain is cultured in a shaking culture mode; the rotation speed of the shaking culture is 90-110 rpm; the time of the shaking culture is 5.5-6.5 hours;

the culture mode of the MS-3S strain is shake culture; the rotation speed of the shaking culture is 90-110 rpm; the time of the shaking culture is 3.5-4.5 hours.

The invention provides a nasal drip vaccine for preventing and controlling coronavirus infection, which comprises the recombinant lactobacillus plantarum and an adjuvant.

The invention provides a fusion gene for expressing coronavirus M protein epitope peptide, which comprises a secretion peptide sequence, a coding coronavirus M protein epitope peptide sequence and an SCWA signal peptide sequence. The secretion peptide and the SCWA signal peptide are respectively added at the two ends of the coronavirus M protein epitope peptide to be expressed, which is beneficial to greatly improving the expression quantity of the coronavirus M protein epitope peptide, simultaneously improving the presentation efficiency of the coronavirus M protein epitope peptide as an antigen, effectively transferring the surface antigen to the antigen presenting cell, enhancing the mucosal immune response aiming at a target site, improving the immunogenicity and being beneficial to the immune system of an organism to play a role. Therefore, the construction of the recombinant lactobacillus vector provided by the invention provides a basis for developing a vaccine for preventing and controlling coronavirus infection.

Furthermore, the invention specifically defines that the novel coronavirus M protein epitope peptide is fused and expressed with MS2 phage capsid protein to form a coding gene sequence of MS2 phage capsid protein embedded with the coronavirus M protein epitope peptide, which is favorable for forming a dimer when the coronavirus M protein epitope peptide is expressed, further effectively transferring surface antigen to antigen presenting cells, enhancing mucosal immune response aiming at target sites and improving immunogenicity.

The invention also provides recombinant lactobacillus plantarum containing the recombinant lactobacillus vector or the recombinant lactobacillus vector obtained by the preparation method. The lactobacillus plantarum is beneficial bacteria, has no endotoxin, adhesion, immune regulation and other characteristics, belongs to safe bacteria without side effects, and simultaneously has the characteristics of safety, high efficiency and induction of organism mucosa immunity by using the vaccine prepared by the lactobacillus. The result of the embodiment of the invention shows that the expression rate of the exogenous gene in the recombinant lactobacillus plantarum prepared by the invention exceeds 30 percent, and the recombinant lactobacillus plantarum has good expression characteristics.

Further, the present invention specifically defines the kind of signal peptide. Experiments prove that different kinds of signal peptides have influence on the expression quantity and immunogenicity of the foreign protein, and the results show that the recombinant lactobacillus plantarum prepared by combining different signal peptides or signal peptides shows green fluorescence, wherein the fluorescence intensity of the recombinant lactobacillus plantarum MS-2S containing the gene sequences for 2145 secretion peptide and SCWA signal peptide is strongest.

Drawings

FIG. 1 is a schematic diagram of PCR amplification products of the novel crown M protein peptide epitope gene of the present invention;

FIG. 2 is a schematic diagram showing the induction expression of the novel crown M protein peptide epitope protein in Lactobacillus plantarum LP18 detected by Western Blot;

FIG. 3 is a schematic representation of the surface expression of immunofluorescence identified recombinant bacteria of the present invention;

FIG. 4 is a schematic representation of recombinant bacterial expression in flow cytometric analysis of the present invention;

FIG. 5 is a schematic diagram of flow cytometry analysis of the effect of rotational speed on recombinant expression in accordance with the present invention;

FIG. 6 is a schematic diagram of Western Blot analysis of the effect of rotational speed on recombinant expression;

FIG. 7 is a schematic diagram of flow cytometry analysis of the effect of initial pH on recombinant expression of the present invention;

FIG. 8 is a schematic diagram of serum ELISA after immunization with recombinant bacteria of the invention;

FIG. 9 is a schematic diagram of a fecal-like ELISA after immunization with recombinant bacteria of the invention;

FIG. 10 is a schematic ELISA of alveolar lavage fluid after immunization with recombinant bacteria of the present invention.

Detailed Description

In the invention, the amino acid sequence of the coronavirus M protein epitope peptide is preferably shown as SEQ ID NO. 27 (ITVATSRT), which is an M protein epitope shared by a novel coronavirus (SARS-CoV-2, NC_045512.2) and a SARS coronavirus (SARS-CoV, NC_ 004718). The encoding coronavirus M protein epitope peptide sequence preferably comprises an encoding gene sequence of MS2 phage capsid protein embedded with coronavirus M protein epitope peptide; MS2 phage capsid protein (MS) chimeric with coronavirus M protein epitope peptide is (ASNFTQFVLVDNGGITVATSRTTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVATQTVGGVELPVAAWRSYLSMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY, SEQ ID NO: 28). The coronavirus M protein epitope peptide and the MS2 phage capsid protein are subjected to fusion expression, and specifically, the coronavirus M protein epitope peptide is placed in a loop structure of the phage capsid protein, so that a dimer is formed during expression, surface antigens can be more effectively transferred to antigen presenting cells, mucosal immune response to target sites is enhanced, immunogenicity is improved, and the immune system of an organism is facilitated to play a role. The result of the embodiment of the invention shows that phage MS2 self-polymerization protein is introduced as a peptide epitope delivery carrier, is expressed in lactic acid bacteria in a recombination mode, can form a dimerization structure, and improves the local peptide epitope concentration.

In the present invention, the fusion gene further includes gene sequences encoding DCpep (SEQ ID NO:29, FYPSYHSTPQRP) and HA tag protein (SEQ ID NO:30, YPYDVPDYA). DCpep functions to promote presentation of peptide epitopes to dendritic cells, exerting immunomodulatory activity; the HA tag protein functions as a tag protein, and imparts a detectable property to the fusion protein, so that the fusion protein not only provides an antigen linear epitope, but also provides a corresponding detection and identification path.

In the present invention, the signal peptide includes 2145 secretory peptide (SEQ ID NO:1, MVKKINLMILGMLVFGVGTGATMINPEMTTAAHASA) or 3050 secretory peptide (SEQ ID NO:2, MEMKKFNFKTMLLLVLASCVVVVVVVNVSLGPQTAITAQASK). The fusion gene preferably includes a 2S fusion gene and a 3S fusion gene depending on the kind of the secretory peptide. The 2S fusion gene is a gene sequence encoding the following fusion protein: 2145 secretion peptide-MS 2 phage capsid protein chimeric with coronavirus M protein epitope peptide-DCpep peptide-SCWA signal peptide-HA tag. The 3S fusion gene is a gene sequence encoding the following fusion protein: 3050 secretion peptide-MS 2 phage capsid protein chimeric with coronavirus M protein epitope peptide-DCpep peptide-SCWA signal peptide-HA tag. Wherein the amino acid sequence of the SCWA signal peptide is shown as SEQ ID NO. 21 (VKPEQPTIPTTPTEPVKPGQLTTPAKPDQPMTSDKSVQTITIKFVGQRLPQTNETDQQHMTLSGLLLLAMSGLLGLLGMAKRQHKE).

In an embodiment of the invention, the peptide (SEQ ID NO:22, the MS2 phage capsid protein embedded with the coronavirus M protein epitope peptide is expressed by combining the 2145 secretion peptide with the SCWA signal peptide, the 3050 secretion peptide with the SCWA signal peptide respectively, and the expression quantity of the exogenous protein on the cell wall is improved, thereby improving the antigen presenting efficiency. In the embodiment of the invention, the recombinant plant lactobacillus vector preferably comprises the following three schemes: a fusion gene comprising a gene sequence encoding a 1320 secretory peptide, a coronavirus M protein epitope peptide and an MS2 bacteriophage capsid protein, a fusion protein comprising a gene sequence encoding a 2145 secretory peptide, an SCWA signal peptide and a coronavirus M protein epitope peptide and an MS2 bacteriophage capsid protein, and a fusion protein comprising a gene sequence encoding a 3050 secretory peptide, an SCWA signal peptide, a coronavirus M protein epitope peptide and an MS2 bacteriophage capsid protein. In the embodiment of the invention, three systems are respectively utilized: 1320 (lipid anchoring system); 2145-SCWA (cell wall anchoring system); 3050-SCWA (cell wall anchoring system) completes the presentation of peptide epitopes. However, the three types of presentation are different, 1320 (lipid anchoring system) is biased to be distributed in a punctiform manner in indirect immunofluorescence research, 2145-SCWA and 3050-SCWA (cell wall anchoring system) show the surface display effect of recombinant protein whole bacteria, and 2145-MS-SCWA has higher expression effect than 3050-MS-SCWA on the same protein although the three types of expression are cell wall anchoring systems.

The invention is not particularly limited in the kind of the skeleton carrier of the recombinant lactobacillus carrier, and skeleton carriers well known in the art can be adopted. In an embodiment of the present invention, the backbone vector comprises a pSIP411 vector.

The invention provides a preparation method of the recombinant lactobacillus vector, which comprises the following steps:

amplifying the sequence of the secretory peptide, the SCWA signal peptide and the coronavirus M protein epitope peptide in the fusion gene respectively;

and (3) cloning the amplified gene sequences encoding the signal peptide, the coronavirus M protein epitope peptide sequence and the SCWA signal peptide into a lactobacillus vector in a connecting way to obtain the recombinant lactobacillus vector.

In an embodiment of the invention, the gene sequence encoding 1320 secretory peptide is shown in SEQ ID NO. 17 (ATGGAGATTTTAGCCATGAATTTCAAAACAGCTGCAAAAGTAACCGTCGTTGCCGGCGCATCCGTACTATTCTTAGCTGCTTGTGGCTCAAGCAAGAGTTCTTCAAGTTCTAAGCAAACGGCCAATTGGACCGAATCAGCCGAATTACCAACGATGGACATTTCTAAGTCCACCGATGTGGTTAGTTCTAACGCGTTGAATAACACCAATGAAGGGTTATACCGTCTCGGTAAGAAC). The primers for amplifying the gene sequence encoding 2145 secretion peptide preferably comprise a forward primer 2145-F01 having a nucleotide sequence shown as SEQ ID NO. 3 and a reverse primer MS-2145-R01 having a nucleotide sequence shown as SEQ ID NO. 4. The gene sequence of the 2145 secretory peptide is shown as SEQ ID NO. 18 (ATGGTGAAAAAAATTAATAAGTTGATGATCTTAGGCATGCTCGTTTTTGGGGTAACGGGGGCAACAATGATCAATCCTGAAATGACGACCGCAGCGCATGCTAGCGCC); the primers for amplifying the gene sequence encoding the 3050 secretion peptide preferably comprise a forward primer sF01 with a nucleotide sequence shown as SEQ ID NO. 5 and a reverse primer MS-3050-R01 with a nucleotide sequence shown as SEQ ID NO. 6. The gene sequence of the 3050 secretory peptide is SEQ ID NO. 19 (ATGGAGATGAAAAAATTTAACTTTAAAACCATGTTGCTATTAGTTTTGGCTAGTTGTGTCTTCGGGGTCGTCGTTAACGTGACTACTAGTCTTGGACCACAAACCGCAATCACCGCCCAGGCCTCCAAG); the primers for amplifying the gene sequence encoding the SCWA signal peptide-HA preferably comprise a forward primer DCpep-SCWA-F01 with a nucleotide sequence shown as SEQ ID NO. 7 and a reverse primer HA-CWA-R01 with a nucleotide sequence shown as SEQ ID NO. 8. The primers for amplifying the fusion gene formed by encoding the coronavirus M protein epitope peptide and the MS2 bacteriophage capsid protein by using the gene sequence of the SCWA signal peptide-HA shown as SEQ ID NO. 20 (GTTAAACCAGAACAACCAACGATACCAACAACACCGACAGAACCGGTTAAACCAGGACAGCTAACGACACCAGCAAAACCAGATCAACCGATGACATCTGATAAGTCGGTTCAAACAATAACCATTAAGTTTGTGGGTCAACGATTACCACAAACTAATGAAACGGATCAACAACATATGACGCTTAGTGGTTTATTGTTATTAGCCATGAGTGGCCTATTAGGTCTCCTTGGTATGGCTAAGCGACAGCACAAAGAGTATCCATATGATGTTCCAGATTATGCTTAA) preferably comprise a forward primer MS-F01 with the nucleotide sequence shown as SEQ ID NO. 9 and a reverse primer SCWA-MS-R01 with the nucleotide sequence shown as SEQ ID NO. 10.

In the present invention, the reaction system for the amplification is preferably 20. Mu.l of KOD One PCR Master Mix enzyme, 1. Mu.l of template, 1. Mu.l of forward primer and 1. Mu.l of reverse primer each at a concentration of 10. Mu.M, and 40. Mu.l of sterile water is filled. The reaction procedure for the amplification is preferably as follows: 98 ℃ for 3min; 15s at 98 ℃, 5s at 60 ℃ and 10s at 72 ℃;35 cycles; 72℃for 5min.

In the present invention, the method of cloning amplified gene sequences encoding a signal peptide, a coronavirus M protein epitope peptide and an MS2 phage capsid protein into a lactic acid bacteria vector preferably comprises the steps of: connecting the amplified gene sequences encoding the signal peptide, the coronavirus M protein epitope peptide and the MS2 phage capsid protein by utilizing overlap PCR to obtain fusion genes encoding the signal peptide, the coronavirus M protein epitope peptide and the MS2 phage capsid protein;

the fusion gene encoding the signal peptide, the coronavirus M protein epitope peptide and the MS2 phage capsid protein is connected into a lactobacillus vector by using a seamless cloning technology.

In the invention, the overlap PCR is completed by using the amplified gene sequence encoding the signal peptide, the gene sequence of the coronavirus M protein epitope peptide and the gene sequence of the MS2 phage capsid protein as templates and using sF01 and SHA-R03 primers. The overlapping PCR method is used for amplifying the same time as the reaction system and the reaction program. Prior to the overlap PCR, the amplified products are preferably subjected to gel recovery for purification purposes.

In the present invention, amplification and sequencing verification are preferably performed after ligation to the lactic acid bacteria vector. The method for amplifying and sequencing verification preferably comprises the steps of electrically converting a connection product into lactococcus for culture, screening and culturing, performing colony PCR detection to obtain recombinant bacteria, sequencing the recombinant bacteria, and extracting plasmids from the recombinant lactobacillus vectors with correct sequencing to obtain a large number of recombinant lactobacillus vectors. The primer for colony PCR detection preferably comprises a forward primer 411test-F01 with a nucleotide sequence shown as SEQ ID NO. 11 and a reverse primer 411test-R01 with a nucleotide sequence shown as SEQ ID NO. 12.

In the present invention, the host cell of the recombinant lactobacillus plantarum is preferably the lactobacillus plantarum LP18 strain. In an embodiment of the invention, the recombinant lactobacillus plantarum preferably comprises an MS-1 strain, an MS-2S strain and an MS-3S strain. The MS-2S strain comprises the 2S fusion gene or a recombinant lactobacillus vector inserted with the 2S fusion gene, and the MS-3S strain comprises the 3S fusion gene or a recombinant lactobacillus vector inserted with the 3S fusion gene.

The method of constructing the recombinant Lactobacillus plantarum is not particularly limited, and may be any method known in the art, such as electrotransformation.

In the embodiment of the invention, three recombinant lactobacillus plantarum are constructed, the expression condition of exogenous genes is detected by indirect immunofluorescence, the expression efficiency of the exogenous genes in MS-2S (comprising 2145 signal peptide and SCWA signal peptide) is highest, the expression efficiency of the exogenous genes in MS-3S (comprising 3050 signal peptide and SCWA signal peptide) is inferior, and the expression efficiency of the exogenous genes in MS-1 (comprising 1320 signal peptide) is lowest.

In the present invention, the method for culturing recombinant lactobacillus plantarum preferably comprises the following steps:

recombinant lactobacillus plantarum is inoculated in MRS liquid culture medium for culture.

In the present invention, the temperature of the culture is preferably 36 to 38℃and more preferably 37 ℃. The culture mode of the MS-1 strain is preferably stationary culture; the stationary culture time is 5.5 to 6.5 hours, more preferably 6 hours. The culture mode of the MS-2S strain is shake culture; the rotation speed of the shaking culture is preferably 90 to 110rpm, more preferably 100rpm; the time of the shaking culture is 5.5 to 6.5 hours, more preferably 6 hours. The culture mode of the MS-3S strain is shake culture; the rotation speed of the shaking culture is preferably 90 to 110rpm, more preferably 100rpm; the time of the shaking culture is preferably 3.5 to 4.5 hours, more preferably 4 hours.

The invention provides application of recombinant lactobacillus plantarum in preparing a nasal drip vaccine for preventing and controlling coronavirus infection.

In the invention, the immunogenicity detection result of the recombinant lactobacillus plantarum shows that the detection of the specific antibodies of the three recombinant bacteria are obviously different, and the three recombinant bacteria have a certain immune effect.

The recombinant lactobacillus vector expressing coronavirus M protein epitope peptide, the recombinant lactobacillus plantarum and the application provided by the invention are described in detail below by combining with examples, but are not to be construed as limiting the scope of the invention.

The primers involved in the examples of the present invention are shown in Table 1:

TABLE 1 primer sequences

Example 1

1. The amplification method of the encoding gene (MS gene) of MS2 phage capsid protein containing coronavirus M protein epitope peptide uses plasmid containing MS gene (SEQ ID NO: 25) synthesized by company as template, adopts MS-F01/SCWA-MS-R01 primer pair to carry out PCR amplification, wherein the reaction system of amplification is: KOD One PCR Master Mix enzyme 20. Mu.l, template 1. Mu.l, MS-F01, SCWA-MS-R01 1. Mu.l each, sterile water make up 40. Mu.l. The reaction procedure is: 98 ℃ for 3min; 15s at 98 ℃, 5s at 60 ℃ and 10s at 72 ℃;35 cycles; 72℃for 5min.

Amplification of 2.2145 secretion peptide encoding genes: the lactobacillus plantarum genome is used as a template, and 2145-F01/MS-2145-R01 primer pair is adopted for PCR amplification, wherein the amplification reaction system is 40 μl.

Amplification of 3.3050 secretion peptide encoding genes: the lactobacillus plantarum genome is used as a template, and sF01/MS-3050-R01 primer pairs are adopted to carry out PCR amplification, wherein the amplification reaction system is 20 mu l of KOD One PCR Master Mix enzyme, 1 mu l of the template, 1 mu l of each sF01 and 1 mu l of each MS-3050-R01, and 40 mu l of sterile water are supplemented. The reaction procedure was 98℃for 3min; 15s at 98 ℃, 5s at 60 ℃ and 10s at 72 ℃;35 cycles; 72℃for 5min.

Amplification of scwa signal peptide encoding gene: the lactobacillus plantarum genome is used as a template, and a DCpep-SCWA-F01/HA-CWA-R01 primer pair is adopted for PCR amplification, wherein the amplification reaction system is 20 mu l of KOD One PCR Master Mix enzyme, 1 mu l of template, 1 mu l of each of DCpep-SCWA-F01 and HA-CWA-R01, and 40 mu l of sterile water are supplemented. The reaction procedure was 98℃for 3min; 15s at 98 ℃, 5s at 60 ℃ and 10s at 72 ℃;35 cycles; 72℃for 5min.

5. And (3) performing agarose gel electrophoresis on each PCR product obtained by amplification, and recovering each band by using a gel recovery kit.

6. Synthesis of the first fusion gene: direct synthesis of the first fusion gene by direct synthesis method comprising 1320, MS, DCpep and HAtag in this order Wherein-> The MS nucleotide sequence is-> Wherein-> Represents a coronavirus M protein epitope peptide);

7. amplification of the second fusion gene: the 2145 secretion peptide coding gene, the SCWA signal peptide coding gene, the coronavirus M protein epitope peptide and the MS2 phage capsid protein coding gene are connected by utilizing an overlap PCR method, and the primer pair for amplification is 2145-F01/HA-CWA-R01. The ligation product is 2145 (BamHI) -MS-DCpep-SCWA-HA sequence of the second fusion gene SEQ ID NO. 15; wherein the wave underline is the MS gene; the italics indicate the M protein epitope peptide gene sequence; the thickened underline is DCpep, and the double underline is double underlineScore SCWA sequence, bold font for HA sequence);

8. amplification of the third fusion gene: the encoding genes of 3050 secretion peptide, SCWA signal peptide (MS gene) and MS2 phage capsid protein containing coronavirus M protein epitope peptide are connected by utilizing an overlapping PCR method, and the primer pair for amplification is sF01/HA-CWA-R01. The ligation product was a third fusion gene encoding 3050 (BamHI) -MS-DCpep-SCWA-HA Wherein the thick underline indicates the 3050 signal peptide gene sequence; the wavy line underlined indicates the MS gene sequence comprising the coronavirus M protein epitope peptide, the bolded underlined indicates the DCpep sequence, the double underlined portion indicates the SCWA sequence-HA sequence, the dotted line portion +.>As a linking sequence).

Example 2

Construction and identification of recombinant lactobacillus vector and recombinant lactobacillus plantarum

The gel recovery products of the three fusion genes prepared in example 1 were respectively connected to pSIP411 vector by seamless cloning technology, electrotransformed into lactococcus NZ3900, spread on GM17 solid plate containing erythromycin (concentration 20. Mu.g/ml), and cultured overnight at 30 ℃; single colonies were picked and tested by colony PCR using 411test-F01/R01, and positive colonies were sequenced after verification. And (3) performing amplification culture on thalli of the expected target fragment obtained by sequencing, and extracting plasmids to obtain recombinant expression plasmids.

Respectively electrotransferring the obtained three recombinant expression plasmids into lactobacillus plantarum LP18, coating on an MRS solid plate containing erythromycin (with the concentration of 20 mu g/ml), and culturing at 37 ℃ overnight; single colonies were picked and colony PCR identified using 411test-F01/R01 universal primers.

The results are shown in FIG. 1. As can be seen from FIG. 1, this example successfully constructed three recombinant Lactobacillus plantarum strains, wherein the engineering strain using 1320 signal peptide was designated MS-1; the engineering strain using 2145 signal peptide and SCWA signal peptide was designated MS-2S; the engineered strain using the 3050 signal peptide and the SCWA signal peptide was designated MS-3S.

Example 3

Inducible expression of recombinant lactobacillus and identification of expression products

MS-1, MS-2S, MS-3S and Lactobacillus plantarum LP18 are inoculated in fresh MRS liquid culture medium (erythromycin concentration is 20 mu g/ml) according to 1% inoculum size respectively, and are subjected to stationary culture at 37 ℃ for 2h, and when OD ₆₀₀ When the value is 0.3-0.5, adding an induced peptide sppip (the concentration is 50 ng/ml), continuing to culture, culturing MS-1 and MS-3S for 4h, culturing MS-2S for 6h, centrifuging at 4000rpm for 6min, collecting recombinant lactobacillus plantarum thalli, flushing 3 times with an equivalent amount of PBS to remove MRS culture medium, re-suspending with an equivalent amount of lysate, grinding with an equivalent amount of glass bead grinder, collecting protein liquid, and preserving at-80 ℃ for standby.

And taking a proper amount of protein liquid expressed by the separated recombinant lactobacillus, adding 5 x SDS-PAGE loading buffer, uniformly mixing, and boiling for 5-8min. After 12% SDS-PAGE electrophoresis, PVDF membrane was transferred by wet transfer for Western Blot analysis: shake-sealing 5% skim milk at room temperature for 2h, and washing with PBST for 3 times; 1:4000 rabbit anti-HA tag antibody 4 ℃ overnight incubation, PBST washing 3 times; incubation of HRP-labeled goat anti-rabbit secondary antibody 1h at 5000, and PBST washing 3 times; ECL development was observed and photographed.

As a result, as shown in FIG. 2, it was found that all three strains expressed the coronavirus M protein peptide epitope protein, were correctly sized, and all showed double bands, i.e., MS-produced dimerization bands.

Example 4

Indirect immunofluorescence validation of exogenous gene expression

Three recombinant Lactobacillus plantarum were inoculated in liquid MRS medium (erythromycin concentration 20. Mu.g/ml) at 1% inoculum size, respectively, and cultured for 2h, and then OD was obtained ₆₀₀ When the value is 0.3-0.5, adding the induced peptide sppip (the concentration is 50 ng/ml), continuing culturing, culturing MS-1 and MS-3S for 4 hours, culturing MS-2S for 6 hours, and collecting the bacterial cells and washing the bacterial cells with the same amount of PBS for 3 times. Blocking with 3% BSA at room temperature for 2h, washing with PBST for 4 times, and washing the primary antibody with anti-HA tag antibody containing rabbit source of 1:100Incubation overnight, PBST wash 3 times, FITC-labeled secondary antibody 1: and (3) incubating for 2h in a dark place 500, washing the PBST for 4 times, tabletting, and observing the expression condition of the exogenous gene under an oil microscope.

As a result, as shown in FIG. 3, compared with the negative control, the surfaces of the three recombinant bacteria all show green fluorescence, wherein the fluorescence intensity of MS-2S is strongest, the fluorescence intensity of MS-3S is weakest, and the fluorescence intensity of MS-1 is weaker.

Example 5

Flow cytometry analysis of exogenous gene expression

1% recombinant Lactobacillus plantarum was inoculated in liquid MRS medium (erythromycin concentration 20. Mu.g/ml) and cultured for 2h, when OD ₆₀₀ When the value is 0.3-0.5, adding an induction peptide sppip (the concentration is 50 ng/ml), continuing to culture, culturing MS-1 and MS-3S for 4 hours, collecting thalli after culturing MS-2S for 6 hours, washing 3 times by using equal amount of PBS, incubating primary antibody with rabbit anti-HA tag antibody containing 1:100 for 2 hours at room temperature, washing 3 times by using PBST, incubating secondary antibody marked by FITC for 2 hours in a dark state 1:500, washing 4 times by using PBST, and taking a proper amount of thalli for analysis by using a flow cytometer.

As a result, as shown in FIG. 4, compared with the negative control, all three recombinant bacteria have an expression rate of more than 30%, wherein the expression rate of MS-2S is highest and is 64.37%; MS-1 expression rate was 39.42%; the expression rate of MS-3S was the lowest and was only 35.93%.

Example 6

High-density culture method of recombinant lactobacillus plantarum

1. The influence of the rotation speed and the initial pH value on the growth and the expression of the recombinant bacteria are respectively studied.

Culturing at different rotation speeds of 0, 50 and 100rpm, and discussing the influence of rotation speed on the growth and expression of lactobacillus. The initial pH of the culture was 5.5.

MS-1 and MS-2S were inoculated in 50ml fresh MRS liquid medium (erythromycin concentration 20. Mu.g/ml) according to an inoculum size of 2%, respectively, and cultured at 37℃for 2 hours at different rotational speeds, when OD ₆₀₀ When the value is 0.3-0.5, adding the induced peptide sppip (the concentration is 50 ng/ml), continuing culturing, culturing for 4h by MS-1, and culturing for 6h by MS-2S. Collecting recombinant Lactobacillus plantarum by centrifuging 5ml culture at 4000rpm for 6min, washing with equal amount of PBS for 3 times to remove MRSThe culture medium was resuspended in an equal amount of lysate, ground with an equal amount of glass bead mill, and the protein liquid was collected and stored at-80℃for further use. Taking a proper amount of protein liquid expressed by the prepared recombinant lactobacillus, adding 5 x SDS-PAGE loading buffer, mixing uniformly, and boiling for 5-8min. After 12% SDS-PAGE electrophoresis, PVDF membrane was transferred by wet transfer for Western Blot analysis: shake-sealing 5% skim milk at room temperature for 2h, and washing with PBST for 3 times; 1:4000 rabbit anti-HA tag antibody 4 ℃ overnight incubation, PBST washing 3 times; incubation of HRP-labeled goat anti-rabbit secondary antibody 1h at 5000, and PBST washing 3 times; ECL development was observed and photographed. And (3) collecting thalli from 5ml of culture, washing 3 times by using an equivalent amount of PBS, incubating the primary antibody with the rabbit anti-HA tag antibody containing 1:100 at room temperature for 2 hours, washing 3 times by using PBST, incubating the secondary antibody marked by FITC for 2 hours in a dark place at 1:500, washing 4 times by using PBST, and taking a proper amount of thalli for analysis by using a flow cytometer.

2. The influence of the initial pH value on the growth and expression of recombinant bacteria was examined, and the initial pH values were adjusted to 5.0, 6.0 and 7.0, respectively, when the culture medium was prepared. The rotation speed of the culture was 100rpm, and the rest steps were the same as above.

The results of flow cytometry analysis of the expression of recombinant Lactobacillus plantarum are shown in FIG. 5, the results of Western Blot analysis of the expression of recombinant Lactobacillus plantarum are shown in FIG. 6, and the results of flow cytometry analysis of the expression of recombinant Lactobacillus plantarum at initial pH are shown in FIG. 7. For MS-1, the expression rate of stationary culture is higher when the initial pH value is 7.0; for MS-2S, an initial pH of 7.0, a rotation of 100rpm resulted in a higher expression rate.

Example 7

Recombinant lactobacillus plantarum immunogenicity detection

1. The experimental groups are shown in Table 2.

Table 2 experimental results

2. Nasal drip immunity

2.1 Experimental materials and instruments

(1) Experimental materials: MRS broth (sterile), 10M NaOH solution, erythromycin, sppip inducing peptide, PBS (sterile), LP18 recombinant strain, pipette tip (sterile), BALB/c mice, 50ml centrifuge tube.

(2) Experimental instrument: a constant temperature incubator at 37 ℃, a constant temperature shaking table, a vertical low-temperature centrifuge and an ultra-clean workbench.

2.2 Experimental procedure

Inoculating recombinant bacteria into 50ml MRS liquid culture medium (erythromycin concentration is 20 mug/ml) with pH of 7.0 according to 2% inoculum size, culturing MS-1 in a constant temperature incubator with temperature of 37 ℃, culturing the other two recombinant bacteria in a constant temperature shaking table (37 ℃ and 100 rpm/min) for 2 hours, adding sppip induction peptide to make the final concentration 100ng/ml, continuing induction culture, standing MS-1 for 4 hours, shaking and inducing MS-2S for 6 hours, and shaking and inducing MS-3S for 4 hours.

Centrifuging the induced bacterial liquid at 4000rpm for 5min, and collecting all recombinant bacteria OD ₆₀₀ The concentration was adjusted so that the 20. Mu.L of the bacterial liquid contained 1X 10 ⁸ The CFU can be used for nasal drip.

2.2.1 immunization schedule:

(1) three days of continuous immunization are performed for the first immunization, feces are taken on the 7 th day, and blood is taken from the eyeorbit on the 8 th day;

(2) second immunization is carried out on the 9 th day, three days of continuous immunization, feces are taken on the 16 th day and the 22 th day, and blood is taken from the eyesockets on the 17 th day and the 23 th day;

(3) performing third immunization on 31 th day, continuously immunizing for three days, collecting feces on 40 th day, collecting blood from the orbit on 41 th day, killing one mouse each group every day on 50 th to 52 th day, respectively collecting feces, serum and alveolar lavage fluid to detect specific antibodies, and collecting spleen and lung separated lymphocytes for flow analysis;

(4) the fourth immunization was performed on day 60, one mouse was not immunized for each fraction, the rest were immunized for all, and the treatment was performed three days, one for each group every day, and feces, serum, and alveolar lavage fluid were taken for detection of specific antibodies, and spleen and lung isolated lymphocytes were taken for flow analysis.

ELISA experiments

3.1 sample treatment:

(1) serum: the mouse blood is kept still for 3 hours at 4 ℃ and at 3500rpm for 5 minutes, light yellow transparent serum is obtained after centrifugation, and the supernatant is lightly absorbed and can be subpackaged in an ultralow temperature refrigerator at-80 ℃ for preservation, and 10 diluents are used.

(2) Faecal content dilution: discarding the first faecal balls, counting from the second, collecting 5 faecal balls from each mouse, weighing, grinding faeces according to 0.1g/300 μl FPBS, centrifuging for 15min at 8000xg, collecting supernatant, and storing in ultralow temperature refrigerator at-80deg.C with 10 dilution.

(3) Alveolar lavage fluid: each mouse is lavaged with 1ml of BPBS, 500 μl of alveolar lavage fluid is recovered, centrifuged at 3000rpm for 5min, and the precipitated cells are removed, and the cells are kept in a sub-packaging at-80deg.C in ultra-low temperature refrigerator with 4-fold dilution.

3.2 antigen coating: the antigen protein was diluted to 2. Mu.g/ml with coating solution, 50. Mu.l of antigen protein dilution was added to each well, and coated overnight at 4 ℃.

3.3 closing: the coated ELISA plate was back-buckled to remove the antigen protein diluent, washed 3 times with PBS, added with 200 μl of 5% skim milk, and blocked at room temperature for 2h.

3.4 primary antibody incubation: PBST was washed 4 times, 200. Mu.l of diluted primary antibody sample (serum, fecal content dilution or alveolar lavage) was added and incubated for 2h at room temperature.

3.5 secondary antibody incubation: PBST was washed 4 times, 200 μl of secondary antibody was added, serum was conjugated with 1:5000 goat anti-mouse HRP conjugated IgG, fecal content was diluted and alveolar lavage fluid was incubated with 1:20000 goat anti-mouse HRP conjugated IgA for 2h at room temperature.

3.6 developing: PBST is washed for 4 times, 100 μl ELISA chromogenic solution is added in a dark place, incubation is carried out for 30min at 37 ℃ in a dark place, the incubation time can be prolonged or shortened appropriately according to the chromogenic condition, and after incubation, 50 μl ELISA stop solution is added to each well to stop the chromogenic reaction.

3.7 detection: immediately after the termination of the reaction, the reaction was detected at a wavelength of 450nm.

As shown in fig. 8-10, compared with the negative control, the specific antibody detection of the three recombinant strains has a significant difference compared with the PBS group, wherein 1320 and 3050-SCWA system induce better humoral immunity after nasal drip, the generation of serum-specific IgG is promoted after polypeptide ELISA verification, 2145-SCWA system can induce stronger specific mucosal immunity after nasal drip, such as alveolar lavage fluid and fecal specific IgA, and the analysis of variance statistics result shows that the immune effect is significant or extremely significant.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. A fusion gene for expressing coronavirus M protein epitope peptide, comprising a secretion peptide sequence, a coronavirus M protein epitope peptide sequence encoding, and an SCWA signal peptide sequence.

2. The recombinant lactic acid bacteria vector according to claim 1, characterized in that said encoding coronavirus M protein epitope peptide sequence comprises a gene sequence encoding MS2 bacteriophage capsid protein chimeric with coronavirus M protein epitope peptide;

the amino acid sequence of the coronavirus M protein epitope peptide is shown as SEQ ID NO. 27;

the signal peptide comprises 2145 secretion peptide or 3050 secretion peptide.

3. The recombinant lactic acid bacteria vector according to claim 2, characterized in that said fusion gene further comprises gene sequences encoding DCpep and HA tag proteins.

4. The recombinant lactic acid bacteria vector according to claim 3, characterized in that said fusion genes comprise 2S fusion genes and 3S fusion genes;

5. A recombinant lactic acid bacteria vector comprising the fusion gene of any one of claims 1 to 4.

6. A recombinant lactobacillus plantarum, characterized in that the recombinant lactobacillus plantarum contains the fusion gene of any one of claims 1 to 4 or the recombinant lactobacillus vector of claim 5.

7. The recombinant lactobacillus plantarum of claim 6, wherein the recombinant lactobacillus plantarum comprises an MS-2S strain and/or an MS-3S strain;

8. The method for culturing recombinant lactobacillus plantarum according to claim 6 or 7, wherein the recombinant lactobacillus plantarum is inoculated in an MRS liquid culture medium for culturing;

the culture temperature is 36-38 ℃;

the initial pH of the culture was 7.0.

9. The method according to claim 8, wherein the MS-2S strain is cultured by shaking culture; the rotation speed of the shaking culture is 90-110 rpm; the time of the shaking culture is 5.5-6.5 hours;

10. A nasal drip vaccine for the prevention and control of coronavirus infection comprising the recombinant lactobacillus plantarum of claim 6 or 7 and an adjuvant.