CN108265073B - Recombinant Bacillus subtilis expressing intestinal M-like cell targeting peptide and porcine epidemic diarrhea virus S protein - Google Patents

Abstract

The invention relates to a recombinant bacillus subtilis (B.S. -RCL) for expressing intestinal M-like cell targeting peptide (L-lectin-beta-GF) and Porcine Epidemic Diarrhea Virus (PEDV) S protein, belonging to the field of biotechnology and genetic engineering. The invention successfully constructs a recombinant bacillus subtilis expression plasmid pHT43-RCL for expressing L-lectin-beta-GF and PEDV S protein, and successfully transfers the recombinant bacillus subtilis expression plasmid into a bacillus subtilis WB800N strain by electric shock. Western-blot verifies that the L-lectin-beta-GF and the PEDV S protein can be successfully expressed in the recombinant bacillus subtilis. In vivo experiments prove that the recombinant bacillus subtilis can effectively target intestinal M-like cells, and oral immunization can stimulate a mouse body to generate a higher-level PEDV specific immune response level. The recombinant bacillus subtilis (B.S. -RCL) is expected to be developed into an enhanced mucosal immune delivery vector and a genetically engineered oral vaccine for preventing porcine epidemic diarrhea.

Description

Recombinant Bacillus subtilis expressing intestinal M-like cell targeting peptide and porcine epidemic diarrhea virus S protein

1. Technical field

The invention relates to recombinant Bacillus subtilis expressing intestinal M-like cell targeting peptide L-lectin-β-GF and PEDV S protein, and belongs to the field of biotechnology genetic engineering. Specifically: Construction of expression plasmid pHT43-RCL for intestinal M-like cell targeting peptide L-lectin-β-GF and PEDVS protein, expression and identification of L-lectin-β-GF and PEDV S protein, recombinant Bacillus subtilis Bacillus can effectively bind to intestinal M-like cells and recombinant Bacillus subtilis can promote the mucosal immunity of mice and the detection of PEDV-specific immune response levels.

2. Background technology

1 Research progress of oral mucosal immunity

Oral mucosal immunization is simple and safe. It not only produces immune responses in local mucosa and other mucosal tissues, but also induces systemic humoral immune responses. It has received close attention from immunologists at home and abroad for many years. The polio sugar pills that are widely taken orally in children at present is an example of successful mucosal immunization of the digestive tract. The application of oral polio vaccine has effectively controlled the incidence of poliomyelitis worldwide and played a guiding role in the development and application of oral vaccine. However, in practice, oral vaccines are often degraded by digestive juices when they pass through the digestive tract, and the vaccines are easily destroyed, which affects the effect of immunity and limits the application and promotion of mucosal immunity in the digestive tract. In recent years, vaccine delivery systems have become one of the hot spots in the research of new technologies in the field of infectious disease defense. In particular, bacterial live vector vaccines have great application potential due to the advantages of convenient culture, large capacity of foreign genes, and strong stimulation of cellular immunity. Bacterial vector vaccine is an important development direction of emerging modern vaccines. The so-called bacterial vector vaccine is to insert the required DNA fragments encoding pathogen-specific antigens into attenuated pathogens or commensal bacteria, so that the encoded antigens can be expressed in order to achieve prevention. the purpose of one or more diseases. The delivery of vaccine antigens through live bacteria carriers can not only stimulate local immune responses in the intestinal tract, but also generate specific immune responses against specific antigens, so that the body can obtain comprehensive protection.

Previous studies have shown that some attenuated bacteria can be used as carriers of vaccine antigens, such as Salmonella, Listeria attenuated and so on. Foreign scholars have successfully expressed the lacZ gene, green fluorescent protein (GFP) gene, HIV-gp140 gene, and Listeria hemolysin gene in eukaryotic cells using attenuated Salmonella as a vector. Salmonella as a vector to deliver DNA vaccines can also be potentially dangerous. First, attenuated bacteria may be in danger of returning to strong virulence; second, plasmid DNA may be integrated into the host cell genome, causing disorder of genes regulating cell growth, uncontrolled expression of proto-oncogenes and tumor suppressor genes, somatic carcinogenesis, A series of problems such as cell transformation. In addition, attenuated bacteria may be potentially pathogenic to some elderly, sick, and disabled individuals. Therefore, choosing an ideal vaccine antigen carrier is the key to establishing new biological techniques. The ideal carrier for delivering antigens should be microorganisms that are both safe to the body and can generate sustained immunity.

2 The effect of Bacillus subtilis on mucosal immunity

Bacterial delivery vehicles for mucosal antigens have been extensively studied. There are many types of live bacterial carriers, and the most widely used include two categories: one is attenuated pathogens that can induce strong and durable immune responses, such as Salmonella and Listeria, but these attenuated bacteria are potentially dangerous as carriers to deliver vaccines , even pathogenic. The other is the safe and non-pathogenic symbiotic species that are currently more concerned, such as probiotics such as Lactobacillus and Bacillus subtilis. As a safe non-pathogenic live bacterial carrier, Bacillus subtilis is a dominant biological population that widely exists in soil and plants. It belongs to the Bacillus family and Bacillus genus. 1.2)μm×(1.5～4.0)μm, single, Gram staining positive, uniform coloring, no capsule, able to move. Bacillus subtilis as an antigen carrier has many advantages: ① It is a probiotic and food additive with human and animal safety records; ② It is low-cost, heat-resistant, anti-drying, easy to transport and use; ③ Its genetic traits and bacteriological levels are easy manipulate. Therefore, Bacillus subtilis is very suitable as a safe oral vaccine and antigen delivery vehicle for other pathogens.

The study found that Bacillus subtilis can be used as a good mucosal immune adjuvant to immunize chicks orally with inactivated avian influenza virus to enhance the non-specific and specific immunity levels of the intestine. Dendritic cells in the piglet gut can take up Bacillus subtilis and migrate to nearby mesenteric lymph nodes to activate T and B lymphocytes and trigger an immune response. Bacillus subtilis as an antigen delivery vehicle can induce mucosal and systemic immune responses, which has attracted great interest from researchers. This provides a broad space for the development of new oral vaccines. But how to further enhance the good mucosal immune carrier of Bacillus subtilis? Many people target another important immune cell on the mucosal surface, M-like cells. Studies have found that Bacillus subtilis can promote an increase in the number of M-like cells in the gut. Intestinal M-like cells are located on the surface of the lymphoid follicles (FAE) of intestinal Peyer's patches, which are pockets and are surrounded by a large number of lymphocytes, such as dendritic cells, T lymphocytes and B lymphocytes. Through M cells, antigens can be more conveniently contacted with antigen-presenting cells or lymphocytes, which can better induce mucosal immune responses.

3 The main structural protein of porcine epidemic diarrhea virus (PEDV) - S protein

Porcine epidemic diarrhea is an acute, contact, and highly contagious enterovirus disease of pigs, characterized by vomiting, watery abdomen, and dehydration in infected pigs. The infection rate of young pigs is as high as 100%, and the mortality rate of infected piglets under 7 days old is as high as 80%-100%. The viral genome is a single positive strand with 7 open reading frames, encoding four structural protein genes, spike glycoprotein (S), membrane glycoprotein (M), nucleocapsid protein (N), small membrane protein (E) ). Among them, the S protein is the surface antigen of PEDV, which plays an important role in regulating the interaction between the host cell receptor protein and the virus, and can receive the virus into the host cell. The most important thing is that the S protein can stimulate the host to produce neutralizing antibodies .

The present invention attempts to express the intestinal M-like cell targeting peptide L-lectin-β-GF and PEDV S protein in the probiotic Bacillus subtilis for M cell targeting effect and oral immunization of live bacteria. It is of great significance to enhance the effect of Bacillus subtilis as a mucosal immune carrier and to open up a new mucosal immune pathway for the prevention of porcine epidemic diarrhea.

3. Content of the Invention

technical problem

The invention successfully constructs the Bacillus subtilis expression plasmid pHT43-RCL of the intestinal M-like cell targeting peptide L-lectin-β-GF and PEDV S protein, and transforms it into the Bacillus subtilis WB800N strain. L-lectin-β-GF and PEDV S proteins were successfully expressed in recombinant Bacillus subtilis WB800N. The recombinant Bacillus subtilis can effectively bind to intestinal M-like cells, which can promote the mucosal immunity and PEDV-specific immune response level of mice. Recombinant Bacillus subtilis strain WB800N (B.S.-RCL) is expected to be an enhanced mucosal immune delivery vehicle and developed as a genetically engineered oral vaccine for the prevention of porcine epidemic diarrhea.

Technical solutions

The invention relates to recombinant Bacillus subtilis (B.S.-RCL) expressing intestinal M-like cell targeting peptide L-lectin-β-GF and PEDV S protein. The recombinant Bacillus subtilis WB800N strain belongs to Bacillus subtilis. Recombinant Bacillus subtilis can effectively bind to intestinal M-like cells to promote mucosal immunity and PEDV-specific immune response in mice.

1 Construction method of expression vector:

1.1 Cloning of the red fluorescent protein gene

Referring to the published red fluorescent protein gene sequence (GenBank accession number: AB166761.1), a pair of primers for amplifying the red fluorescent protein gene RFP was designed, and the 5' end of the upstream primer was introduced with the pHT43 vector multi-cloning site 3' end 11bp Homology arm sequence, BamHI restriction site is introduced between the two sequences (underlined is restriction site); 12bp homology with the 5' end of porcine epidemic diarrhea virus (PEDV) S protein gene is introduced at the 5' end of the downstream primer Arm sequence, BamHI restriction site is introduced between the two sequences (underline is restriction site), and the primer sequence is as follows:

P1: CATCARCCGTA GGATCC AGTAAAGGAGAAGAA (SEQ ID NO. 4);

P2: AACAAAAGAAAT GGATCC TTTGTATAGTTCATCCAT (SEQ ID NO. 5);

PCR amplification was carried out with pDsRed-monomer-N1 plasmid as template; PCR reaction system was 50 μL, PCR reaction conditions: pre-denaturation at 95 °C for 5 min, 95 °C for 30 sec, 72 °C for 30 sec, 72 °C for 1 min, 30 cycles, 72 °C for 10 min, 4 °C 10min; the PCR product was connected to the pMD19-T vector, the constructed vector was named T-R, and the red fluorescent protein gene sequence SEQ ID NO.1 with the complete coding region was obtained after sequencing;

1.2 Cloning of porcine epidemic diarrhea virus (PEDV) S protein gene

Referring to the published porcine epidemic diarrhea virus S protein gene sequence (GenBank accession number: JX560761.1), a pair of primers were designed to amplify the S protein gene cDNA, and the 5' end of the upstream primer was introduced with the 3' end of the red fluorescent protein gene 12bp The homology arm sequence of the two sequences, the BamHI restriction site is introduced between the two sequences (the underline is the restriction site); the 5' end of the downstream primer is introduced with the intestinal M cell targeting peptide L-lectin-β-GF gene 5' The homology arm sequence of the terminal 20bp, the primer sequence is as follows:

P3: GAACTATACAAA GGATCC ATTTCTTTTGTT (SEQ ID NO. 6);

P4: GTCGTCRCTRCTGACRCAAATTTATCATCATCATCTTTAT (SEQ ID NO. 7);

The total RNA extracted from the SD-M strain of porcine epidemic diarrhea virus was used as a template, and the first strand of cDNA was synthesized by MLV-reverse transcriptase and then amplified by PCR; the PCR reaction system was 50 μL, and the PCR reaction conditions: pre-denaturation at 95°C 5min, 95℃ for 30sec, 67℃ for 30sec, 72℃ for 1min, 30cycles, 72℃ for 10min, 4℃ for 10min; the PCR product was ligated to the pMD19-T vector, and the constructed vector was named T-COE. The porcine epidemic diarrhea virus S protein gene sequence SEQID NO.2 of the region;

1.3 Cloning of intestinal M-like cell targeting peptide L-lectin-β-GF gene

Referring to the published L-lectin-β-GF gene sequence (GenBank accession number: BA000018.3), a pair of primers for amplifying the L-lectin-β-GF gene were designed, and the 5' end of the upstream primer was introduced with porcine epidemic The 20 bp homology arm sequence at the 3' end of the diarrhea virus S protein gene; the 5' end of the downstream primer introduces a 15 bp homology arm sequence with the 5' end of the pHT43 multi-cloning site, and an XmaI restriction site is introduced between the two sequences (underlined as enzyme cleavage site), the primer sequences are as follows:

P5: ATAAAGATGATGATGATAAATTTRCGTCARCARCGACGAC (SEQ ID NO. 8); P6: CATTAGRCGGRCTRC CCCGGG AGTAAAATAATATGT (SEQ ID NO. 9);

The genome extracted from Staphylococcus aureus strains was used as a template for PCR amplification; the PCR reaction system was 50 μL, and the PCR reaction conditions were: 95℃ for 5min, 95℃ for 30sec, 69℃ for 30sec, 72℃ for 2min, 30cycles, 72℃ 10min at 4°C for 10min; the PCR product was ligated to the pMD19-T vector with T4 ligase, and the constructed vectors were named T-L; after sequencing, the L-lectin-β-GF gene sequence SEQ ID NO.3 was obtained;

1.4 Construction of recombinant expression vector pHT43-RCL

Take SEQ ID NO.1 sequence, SEQ ID NO.2 sequence and SEQ ID NO.3 sequence as templates, use primers P1, P4 to carry out overlapping PCR amplification, the PCR reaction system is 50 μL, PCR reaction conditions: 95 ℃ pre-denaturation 5min , 95℃ for 30sec, 72℃ for 30sec, 72℃ for 4min, 30cycles, 72℃ for 10min, 4℃ for 10min; the PCR product was recovered and cloned into the pHT43 vector using the One Step Cloning Kit site-directed cloning kit. After sequencing, the expression vector pHT43- RCL.

2 Transformation methods of recombinant plasmids:

Take 60 μL of Bacillus subtilis WB800N electrotransformed competent cells and 1 μL (50 ng/μL) recombinant expression vector pHT43-RCL plasmid mixed into the electric shock cup, and electric shock was performed after ice bath for 5 minutes. Electric shock conditions: 22KV/cm, 25 μF, 200Ω, electric shock once. Add 1 mL of electroshock recovery solution, incubate at 37°C at 100 rpm for 3 h, spread on LB plate of chloramphenicol-resistant solid basal medium, and see positive colonies within 14-18 h.

3. Validation method of protein expression:

Normal Bacillus subtilis WB800N strain (without foreign genes in genetic material) (B.S.) and recombinant Bacillus subtilis WB800N strain expressing intestinal M-like cell targeting peptide L-lectin-β-GF and PEDV S protein (B.S.- RCL) was treated with lysozyme, and the expression products were subjected to Western-blot analysis. After SDS electrophoresis, protein transfer was performed; the transferred PVDF membrane was blocked with 5% skim milk, and then incubated with mouse anti-RFP antibody overnight; after washing, appropriately diluted horseradish peroxidase-labeled rabbit anti-mouse IgG was added at 25°C After 2 hours of action, ECL chemiluminescent solution was exposed and photographed.

4 Recombinant Bacillus subtilis targeting intestinal M-like cells detection method:

Normal Bacillus subtilis WB800N strain (without foreign genes in genetic material) (B.S.), control recombinant Bacillus subtilis WB800N strain (expressing PEDV S protein, but not expressing intestinal M-like cell targeting peptide L-lectin-β- GF) (B.S.-RC) and recombinant Bacillus subtilis WB800N strain (B.S.-RCL) expressing intestinal M-like cell targeting peptides L-lectin-β-GF and PEDV S protein, respectively, were labeled with red fluorescent dye and intestinal In situ perfusion test and immunofluorescence staining were used to observe the binding of recombinant Bacillus subtilis to intestinal M-like cells. The specific method is as follows: after 4-week-old mice were anesthetized with chloral hydrate, the abdominal cavity was opened, and the intestinal tract containing Peyer's patches was ligated in multiple segments, and B.S, B.S.-RC or B.S.-RCL was injected into each segment; 15min and 1h later , the isolated intestinal segments were fixed in 4% paraformaldehyde and embedded with OTC. The embedded intestinal tissue was frozen sectioned and M cells were labeled with UEA-1, followed by confocal microscopy.

5. Detect the level of PEDV-specific immune response produced by oral immunization of the recombinant Bacillus subtilis mice:

Normal Bacillus subtilis WB800N strain (without foreign genes in the genetic material) (BS), control recombinant Bacillus subtilis WB800N strain (BS-RC) and expression of intestinal M-like cell targeting peptide L-lectin-β-GF and The recombinant Bacillus subtilis WB800N strain of PEDV S protein (BS-RCL) was orally immunized to 6-week-old mice at an oral dose of 10 ¹⁰ cfu/kg. The second and third immunizations were performed at the same dose after the first immunization for 7 days and 14 days. Serum and intestinal rinses were collected every week, and the feeding cycle was 35 days. The level of PEDV-specific IgG and specific SIgA was detected by indirect ELISA. The detection method was as follows: PEDV antigen was coated overnight, blocked with 1% BSA (bovine serum albumin) at 37°C for 2h, and then added to the sample to be tested at 37°C after washing. Act for 1h, wash and add appropriately diluted horseradish peroxidase-labeled rabbit anti-mouse IgG or IgA antibody, 37℃ for 1h, TMB chromogenic solution containing tetramethylbenzidine and H ₂ O ₂ after color development Detection of _OD450 . The results showed that the recombinant Bacillus subtilis (BS-RCL) expressing the intestinal M cell targeting peptide L-lectin-β-GF and PEDV S protein can be more significantly than the control recombinant Bacillus subtilis WB800N strain (BS-RC). Stimulate the mouse body to produce an effective level of PEDV-specific immune response.

beneficial effect

1 The expression plasmid pHT43-RCL constructed in this experiment is the first recombinant Bacillus subtilis expression plasmid expressing intestinal M-like cell targeting peptide L-lectin-β-GF and porcine epidemic diarrhea virus S protein at home and abroad.

2. The present invention successfully constructed the intestinal M-like cell targeting peptide L-lectin-β-GF and porcine epidemic diarrhea virus S protein recombinant Bacillus subtilis expression plasmid pHT43-RCL, and transformed into Bacillus subtilis WB800N. It was verified by Western-blot that L-lectin-β-GF and PEDV S proteins were successfully expressed in recombinant Bacillus subtilis WB800N. The recombinant Bacillus subtilis can effectively bind to intestinal M-like cells, and can also promote the production of PEDV-specific immune response levels in mice. Recombinant Bacillus subtilis strain WB800N (B.S.-RCL) is expected to be an enhanced mucosal immune delivery vehicle and developed as a genetically engineered oral vaccine for the prevention of porcine epidemic diarrhea.

3. The present invention constructs a Bacillus subtilis delivery and expression system, which can insert various antigens, active peptides and drugs, etc., which is beneficial to the development of various recombinant Bacillus subtilis vaccines, and is used for Bacillus subtilis in animal mucosal immunity and oral genetic engineering live vaccines, etc. groundwork for research. The present invention will be further described below with reference to the accompanying drawings and specific implementation techniques.

Description of drawings

Figure 1: Schematic diagram of the constructed expression vector plasmid pHT43-RCL

Figure 2: Western-blot image of recombinant Bacillus subtilis WB800N expression product

Figure 3: Recombinant Bacillus subtilis WB800N binding to intestinal M-like cells

Figure 4: Effect of recombinant Bacillus subtilis WB800N (B.S.-RCL) on changes in PEDV-specific IgG levels in mouse serum

Figure 5: Effect of recombinant Bacillus subtilis WB800N (B.S.-RCL) on changes in PEDV-specific SIgA levels in mouse intestinal washes

Detailed ways

1 Construction of expression plasmid pHT43-RCL

The methods used in the following examples are conventional methods unless otherwise specified. The specific materials and reagents involved are as follows: Bacillus subtilis WB800N strain and Staphylococcus aureus strain were donated by Gao Xuewen, School of Plant Protection, Nanjing Agricultural University; Porcine Epidemic Diarrhea Virus SD-M strain was donated by He Kongwang, a researcher at the Veterinary Institute of Jiangsu Academy of Agricultural Sciences. E.coliJM109 strain and plasmid pMD19-T were purchased from Takara company; plasmid pHT43 was purchased from Life Technologies company; plasmid pDsRed-monomer-N1 was purchased from Clontech company; reagent: Q5 high fidelity enzyme was purchased from NEB company; agarose gel recovery Kits, T4 ligase, Trizol, RNA reverse transcription kits and One Step Cloning Kit site-directed cloning kits were purchased from Takara; chloramphenicol, ampicillin, bacterial plasmid extraction kits and bacterial genome extraction kits were all purchased from Takara. Purchased from OMEGA company; mouse anti-RFP antibody, horseradish peroxidase-labeled rabbit anti-mouse IgG and IgA antibody were purchased from BETHYL company; TMB chromogenic solution was purchased from TIANGEN company; PVDF membrane, skim milk and ECL chemiluminescent solution were Purchased from Merck Millipore Corporation. Red fluorescent dye, BSA, chloral hydrate and UEA-1 were purchased from Sigma Company; OTC was purchased from Thermo Company.

1.1 Cloning of the red fluorescent protein gene

Referring to the published red fluorescent protein gene sequence (GenBank accession number: AB166761.1), a pair of primers for amplifying the red fluorescent protein gene RFP was designed, and at the 5 end of the upstream primer, the 11bp identical to the 3' end of the pHT43 vector multi-cloning site was introduced. Source arm sequence, BamHI restriction site is introduced between the two sequences (underlined is restriction site); at the 5 end of the downstream primer, a homology arm sequence with 12 bp at the 5' end of porcine epidemic diarrhea virus (PEDV) S protein gene is introduced , BamHI restriction site (underlined is restriction site) is introduced between the two sequences, and the primer sequence is as follows:

P1: CATCARCCGTA GGATCC AGTAAAGGAGAAGAA

P2: AACAAAAGAAAT GGATCC TTTGTATAGTTCATCCAT

The above primers were synthesized by Shanghai Yingweijieji Trading Co., Ltd.;

PCR amplification was carried out with pDsRed-monomer-N1 plasmid as template; PCR reaction system was 50 μL, PCR reaction conditions: pre-denaturation at 95 °C for 5 min, 95 °C for 30 sec, 72 °C for 30 sec, 72 °C for 1 min, 30 cycles, 72 °C for 10 min, 4 °C 10min; excised the target band, recovered by DNA purification kit, ligated to pMD19-T vector with T4 ligase, the constructed vector was named T-R, and sequenced after colony PCR and enzyme digestion verification. The obtained sequences were aligned with those published in GenBank by BLAST and DNAstar. The cloned red fluorescent protein gene sequence SEQ ID NO. 1 has 100% homology with the sequence published in GenBank.

1.2 Cloning of porcine epidemic diarrhea virus (PEDV) S protein gene

Referring to the published porcine epidemic diarrhea virus S protein gene sequence (GenBank accession number: JX560761.1), a pair of primers for amplifying the S protein gene cDNA was designed, and the 5 end of the upstream primer was introduced into the 12bp of the 3' end of the red fluorescent protein gene Homologous arm sequence, BamHI restriction site is introduced between the two sequences (underlined is restriction site); 20bp at the 5' end of the intestinal M cell targeting peptide L-lectin-β-GF gene is introduced at the 5 end of the downstream primer The homology arm sequence of , the primer sequence is as follows:

P3: GAACTATACAAA GGATCC ATTTCTTTTGTT

P4: GTCGTCRCTRCTGACRCAAATTTATCATCATCATCTTTAT

The above primers were synthesized by Shanghai Yingweijieji Trading Co., Ltd.;

The total RNA extracted from the SD-M strain of porcine epidemic diarrhea virus was used as a template, and the first strand of cDNA was synthesized by MLV-reverse transcriptase and then amplified by PCR; the PCR reaction system was 50 μL, and the PCR reaction conditions: pre-denaturation at 95°C 5min, 95°C for 30sec, 67°C for 30sec, 72°C for 1min, 30cycles, 72°C for 10min, 4°C for 10min; excised the target band, recovered by DNA purification kit, and ligated to pMD19-T vector with T4 ligase to construct The vectors were named T-COE respectively, and were sequenced after colony PCR and enzyme digestion verification. The obtained sequences were aligned with those published in GenBank by BLAST and DNAstar. The cloned porcine epidemic diarrhea virus S protein gene sequence SEQ ID NO. 2 has 100% homology with the sequence published in GenBank.

1.3 Cloning of intestinal M-like cell targeting peptide L-lectin-β-GF gene

Referring to the published L-lectin-β-GF gene sequence (GenBank accession number: BA000018.3), a pair of primers for amplifying the L-lectin-β-GF gene was designed, and the 5 end of the upstream primer was introduced with porcine epidemic diarrhea The 20bp homology arm sequence at the 3' end of the viral S protein gene; the 5 end of the downstream primer introduces a 15bp homology arm sequence with the 5' end of the pHT43 multi-cloning site, and an XmaI restriction site is introduced between the two sequences (underlined is restriction enzyme cut) site), the primer sequences are as follows:

P5: ATAAAGATGATGATGATAAATTTRCGTCARCARCGACGAC

P6: CATTAGRCGGRCTRC CCCGGG AGTAAAATAATATGT

The above primers were synthesized by Shanghai Yingweijieji Trading Co., Ltd.;

The genome extracted from Staphylococcus aureus strains was used as a template for PCR amplification; the PCR reaction system was 50 μL, and the PCR reaction conditions were: 95℃ for 5min, 95℃ for 30sec, 69℃ for 30sec, 72℃ for 2min, 30cycles, 72℃ 10min at 4°C for 10min; excised the target band, recovered by DNA purification kit, ligated to the pMD19-T vector with T4 ligase, the constructed vector was named T-L, and sequenced after colony PCR and enzyme digestion verification. The obtained sequences were aligned with those published in GenBank by BLAST and DNAstar. The cloned intestinal M-like cell targeting peptide L-lectin-β-GF gene sequence SEQ ID NO.3 has 100% homology with the sequence published in GenBank.

1.4 Construction of recombinant expression vector pHT43-RCL

Take SEQ ID NO.1 sequence, SEQ ID NO.2 sequence and SEQ ID NO.3 sequence as templates, use primers P1, P4 to carry out overlapping PCR amplification, the PCR reaction system is 50 μL, PCR reaction conditions: 95 ℃ pre-denaturation 5min , 95°C for 30sec, 72°C for 30sec, 72°C for 4min, 30cycles, 72°C for 10min, 4°C for 10min; as shown in Figure 1, the PCR product was recovered and cloned into pHT43 vector using the OneStep Cloning Kit site-directed cloning kit to obtain Expression vector pHT43-RCL.

2 Transformation methods of recombinant plasmids:

Take 60 μL of Bacillus subtilis WB800N electrotransformed competent cells and 1 μL (50 ng/μL) recombinant expression vector pHT43-RCL plasmid mixed into the electric shock cup, and electric shock was performed after ice bath for 5 minutes. Electric shock conditions: 22KV/cm, 25 μF, 200Ω, electric shock once. Add 1 mL of electroshock recovery solution, incubate at 37°C at 100 rpm for 3 h, spread on LB plate of chloramphenicol-resistant solid basal medium, and see positive colonies within 14-18 h.

3. Validation method of protein expression:

Bacillus subtilis WB800N was treated with lysozyme, and the expression product was analyzed by Western-blot. After SDS electrophoresis, protein transfer was performed; the transferred PVDF membrane was blocked with 5% skim milk, and then incubated with mouse anti-GFP antibody overnight; after washing, appropriate diluted horseradish peroxidase-labeled rabbit anti-mouse IgG was added at 25°C After 2 hours of action, ECL chemiluminescent solution was exposed and photographed. The results showed a positive reaction with the RFP antibody, as shown in Figure 2: 4, 5, and 6 were recombinant Bacillus subtilis WB800N expressing L-lectin-β-GF and PEDVS proteins, and 1, 2, and 3 were normal Bacillus subtilis WB800N (genetic There is an obvious western blot band at 95kD, and no non-specific band appears, which proves that L-lectin-β-GF and PEDV S protein can be in the recombinant Bacillus subtilis WB800N been successfully expressed.

4 Recombinant Bacillus subtilis targeting intestinal M-like cells detection method:

Normal Bacillus subtilis WB800N strain (without foreign genes in genetic material) (B.S.), control recombinant Bacillus subtilis WB800N strain (expressing PEDV S protein, but not expressing intestinal M-like cell targeting peptide L-lectin-β- GF) (B.S.-RC) and recombinant Bacillus subtilis WB800N strain (B.S.-RCL) expressing intestinal M-like cell targeting peptides L-lectin-β-GF and PEDV S protein, respectively, were labeled with red fluorescent dye and intestinal In situ perfusion test and immunofluorescence staining were used to observe the binding of recombinant Bacillus subtilis to intestinal M-like cells. The specific method is as follows: after 4-week-old mice were anesthetized with chloral hydrate, the abdominal cavity was opened, and the intestinal tract containing Peyer's patches was ligated in multiple segments, and B.S, B.S.-RC or B.S.-RCL was injected into each segment; 15min and 1h later , the isolated intestinal segments were fixed in 4% paraformaldehyde and embedded with OTC. The embedded intestinal tissue was frozen sectioned and M cells were labeled with UEA-1, followed by confocal microscopy. As shown in Figure 3: 15min after intestinal ligation, B.S.-RCL and M cells showed obvious co-localization phenomenon; 1h later, the amount of recombinant Bacillus subtilis in Peyer's patches of intestinal segment perfused with B.S.-RCL was significantly increased.

5. Detect the level of PEDV-specific immune response produced by oral immunization of the recombinant Bacillus subtilis mice:

Normal Bacillus subtilis WB800N strain (without foreign genes in the genetic material) (BS), control recombinant Bacillus subtilis WB800N strain (BS-RC) and expression of intestinal M-like cell targeting peptide L-lectin-β-GF and The recombinant Bacillus subtilis WB800N strain of PEDV S protein (BS-RCL) was orally immunized to 6-week-old mice at an oral dose of 10 ¹⁰ cfu/kg. The second and third immunizations were performed at the same dose after the first immunization for 7 days and 14 days. Serum and intestinal rinses were collected every week, and the feeding cycle was 35 days. The level of PEDV-specific IgG and specific SIgA was detected by indirect ELISA. The detection method was as follows: PEDV antigen was coated overnight, blocked with 1% BSA (bovine serum albumin) at 37°C for 2h, and then added to the sample to be tested at 37°C after washing. Act for 1h, wash and add appropriately diluted horseradish peroxidase-labeled rabbit anti-mouse IgG or IgA antibody, 37℃ for 1h, TMB chromogenic solution containing tetramethylbenzidine and H ₂ O ₂ after color development Detection of _OD450 . The final data were calculated by the S/N method. As shown in Figure 4: Oral immunization with recombinant Bacillus subtilis WB800N (BS-RC and BS-RCL), 14 days after the first immunization, the PEDV-specific antibody in serum was significantly higher than that of PBS group and BS group (P<0.01 or P<0.05) , the antibody level remained stable until the 28th day, and then decreased; although there was no significant difference between the BS-RCL group and the BS-RC group, there was an obvious upward trend. Figure 5 shows that: after oral immunization of recombinant Bacillus subtilis WB800N (BS-RC and BS-RCL), 14 days after the first immunization, the serum PEDV-specific antibody was significantly higher than that of the PBS group and the BS group (P<0.01 or P<0.05). After that, the antibody level remained stable until the 21st day, and then decreased; although there was no significant difference between the BS-RCL group and the BS-RC group, there was an obvious upward trend.

6 Data Analysis

One-way ANOVA variance test results of SPSS 17.0 software were used, and statistical analysis was performed on the test data. The statistical results were expressed as mean ± standard error (Mean ± SE). The criterion for the significance of difference is: P<0.05, significant difference (*); P<0.01, extremely significant difference (**).

application

The present invention relates to a recombinant Bacillus subtilis WB800N strain (B.S.-RCL) expressing intestinal M-like cell targeting peptide L-lectin-β-GF and PEDV S protein, which is expected to be an enhanced mucosal immune delivery carrier and developed to prevent swine epidemics A genetically engineered oral vaccine for diarrhoea; at the same time, it provides a technical solution for the study of Bacillus subtilis mucosal immune delivery live vector.

sequence listing

<110> Nanjing Agricultural University

<120> Recombinant Bacillus subtilis expressing intestinal M-like cell targeting peptide and porcine epidemic diarrhea virus S protein

<140> 2017100121404

<141> 2017-01-03

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 714

<212> DNA

<213> DsRed gene sequence (Artificial Sequence)

<400> 1

atgagtaaag gagaagaact tttcactgga gttgtcccaa ttcttgttga attagatggt 60

gatgttaatg ggcacaaatt ttctgtcagt ggagagggtg aaggtgatgc aacatacgga 120

aaacttaccc ttaaatttat ttgcactact ggaaaactac ctgttccatg gccaacactt 180

gtcactactt tcggttatgg tgttcaatgc tttgcgagat acccagatca tatgaaacag 240

catgactttt tcaagagtgc catgcccgaa ggttatgtac aggaaagaac tatatttttc 300

aaagatgacg ggaactacaa gacacgtgct gaagtcaagt ttgaaggtga tacccttgtt 360

aatagaatcg agttaaaagg tattgatttt aaagaagatg gaaacattct tggacacaaa 420

ttggaataca actataactc acacaatgta tacatcatgg cagacaaaca aaagaatgga 480

atcaaagtta acttcaaaat tagacacaac attgaagatg gaagcgttca actagcagac 540

cattatcaac aaaatactcc aattggcgat ggccctgtcc ttttaccaga caaccattac 600

ctgtccacac aatctgccct ttcgaaagat cccaacgaaa agagagacca catggtcctt 660

cttgagtttg taacagctgc tgggattaca catggcatgg atgaactata caaa 714

<210> 2

<211> 876

<212> DNA

<213> Porcine epidemic diarrhea virus S protein gene sequence (Artificial Sequence)

<400> 2

atgatttctt ttgttactcc gccatcattt aatgaccatt cttttgttaa cattactgtc 60

tctgcttcct ttggtggtca tagtggtgcc aaccttattg catctgacac tactatcaat 120

gggtttagtt ctttctgtgt tgacactaga caatttacca tttcactgtt ttacaacgtt 180

acaaacagtt atggttatgt gtctaaatca caggacagta attgcccttt caccttgcaa 240

tctgttaatg attacctgtc ttttagcaaa ttttgtgttt ccaccagcct tttggctagt 300

gcctgtacca tagatctttt tggttaccct gagtttggta gtggtgttaa gtttacgtcc 360

ctttactttc aattcacaaa gggtgagttg attactggca cgcctaaacc acttgaaggt 420

gtcacggacg tttcttttat gactctggat gtgtgtacca agtatactat ctatggcttt 480

aaaggtgagg gtatcattac ccttacaaat tctagctttt tggcaggtgt ttattacaca 540

tctgattctg gacagttgtt agcctttaag aatgtcacta gtggtgctgt ttattctgtt 600

acgccatgtt ctttttcaga gcaggctgca tatgttgatg atgatatagt gggtgttatt 660

tctagtttgt ctagctccac ttttaacagt actagggagt tgcctggttt cttctaccat 720

tctaatgatg gctctaattg tacagagcct gtgttggtgt atagtaacat aggtgtttgt 780

aaatctggca gtattggcta cgtcccatcc cagtctggcc aagtcaagac tgcacccacg 840

gttactggga atattagtat tcccaccaac tttagt 876

<210> 3

<211> 987

<212> DNA

<213> L-lectin-β-GF gene sequence (Artificial Sequence)

<400> 3

atgtttgcgt cagcagcgac gacaaccgca gtaactgcta atacaattac agttaataaa 60

gataacttaa aacaatatat gacaacgtca ggtaatgcta cctatgatca aagtaccggt 120

attgtgacgt taacacagga tgcatacagc caaaaaggtg ctattacatt aggaacacgt 180

attgactcta ataagagttt tcatttttct ggaaaagtaa atttaggtaa caaatatgaa 240

gggcatggaa atggtggaga tggtatcggt tttgcctttt caccaggtgt attaggtgaa 300

acagggttaa acggtgccgc agtaggtatt ggtggcttaa gtaacgcatt tggcttcaaa 360

ttggatacgt atcacaatac atctaaacca aattcagctg caaaggcgaa tgctgaccca 420

tctaatgtag ctggtggagg tgcgtttggt gcatttgtaa caacagatag ttatggtgtt 480

gcgacaacgt atacatcaag ttcaacagct gataatgctg cgaagttaaa tgttcaacct 540

acaaataaca cgttccaaga ttttgatatt aactataatg gtgatacaaa ggttatgact 600

gtcaaatatg caggtcaaac atggacacgt aatatttcag attggattgc gaaaagtggt 660

acgaccaact tttcattatc aatgacagcc tcaacaggtg gcgcgacaaa tttacaacaa 720

gtacaatttg gaacattcga atatacagag tctgctgtta cacaagtgag atacgttgat 780

gtaacaacag gtaaagatat tattccacca aaaacatatt caggaaatgt tgatcaagtc 840

gtgacaatcg ataatcagca atctgcattg actgctaaag gatataacta cacgtccgtc 900

gatagttcat atgcgtcaac ttataatgat acaaataaaa ctgtaaaaat gacgaatgct 960

ggacaatcag tgacatatta ttttact 987

<210> 4

<211> 32

<212> DNA

<213> P1 primer (Artificial Sequence)

<400> 4

catcarccgt aggatccagt aaaggagaag aa 32

<210> 5

<211> 36

<212> DNA

<213> P2 primer (Artificial Sequence)

<400> 5

aacaaaagaa atggatcctt tgtatagttc atccat 36

<210> 6

<211> 30

<212> DNA

<213> P3 primer (Artificial Sequence)

<400> 6

gaactataca aaggatccat ttcttttgtt 30

<210> 7

<211> 40

<212> DNA

<213> P4 primer (Artificial Sequence)

<400> 7

gtcgtcrctr ctgacrcaaa tttatcatca tcatctttat 40

<210> 8

<211> 40

<212> DNA

<213> P5 primer (Artificial Sequence)

<400> 8

ataaagatga tgatgataaa tttrcgtcar carcgacgac 40

<210> 9

<211> 36

<212> DNA

<213> P6 primer (Artificial Sequence)

<400> 9

cattagrcgg rctrccccgg gagtaaaata atatgt 36

Claims (3)

1. a recombinant expression plasmid pHT43-RCL of intestinal M-like cell targeting peptide L-lectin-β-GF and PEDV S protein, is characterized in that, described recombinant expression plasmid pHT43-RCL is constructed by the following method : 1.1 Cloning of the red fluorescent protein gene Design a pair of primers to amplify the red fluorescent protein gene RFP with reference to the published red fluorescent protein gene sequence, the GenBank accession number of the red fluorescent protein gene sequence is AB166761.1, and introduce polyclonal with pHT43 vector at the 5' end of the upstream primer The 11 bp homology arm sequence at the 3' end of the site, the BamHI restriction site is introduced between the two sequences, and the underline is the restriction site; the 5' end of the porcine epidemic diarrhea virus S protein gene is introduced at the 5' end of the downstream primer. The homology arm sequence of 12bp, the BamHI restriction site is introduced between the two sequences, and the underline is the restriction site, and the primer sequence is as follows: P1: CATCARCCGTA GGATCC AGTAAAGGAGAAGAA, sequence number is SEQ ID NO.4; P2: AACAAAAGAAAT GGATCC TTTGTATAGTTCATCCAT, sequence number is SEQ ID NO.5; PCR amplification was carried out with pDsRed-monomer-N1 plasmid as template; PCR reaction system was 50 μL, PCR reaction conditions: pre-denaturation at 95 °C for 5 min, 95 °C for 30 sec, 72 °C for 30 sec, 72 °C for 1 min, 30 cycles, 72 °C for 10 min, 4 °C 10min; the PCR product was connected to the pMD19-T vector, the constructed vector was named T-R, and the red fluorescent protein gene sequence SEQ ID NO.1 with the complete coding region was obtained after sequencing; 1.2 Cloning of porcine epidemic diarrhea virus S protein gene Design a pair of primers for amplifying S protein gene cDNA with reference to the published porcine epidemic diarrhea virus S protein gene sequence. The GenBank accession number of the porcine epidemic diarrhea virus S protein gene sequence is JX560761.1, and the 5' of the upstream primer is The 12bp homology arm sequence with the 3' end of the red fluorescent protein gene was introduced at the end, and a BamHI restriction site was introduced between the two sequences, of which the underline was the restriction site; the 5' end of the downstream primer was introduced to target intestinal M cells The homology arm sequence of 20bp at the 5' end of the peptide L-lectin-β-GF gene, the primer sequence is as follows: P3: GAACTATACAAA GGATCC ATTTCTTTTGTT, sequence number is SEQ ID NO.6; P4: GTCGTCRCTRCTGACRCAAATTTATCATCATCATCTTTAT, sequence number is SEQ ID NO.7; The total RNA extracted from the SD-M strain of porcine epidemic diarrhea virus was used as a template, and the first strand of cDNA was synthesized by MLV-reverse transcriptase and then amplified by PCR; the PCR reaction system was 50 μL, and the PCR reaction conditions: pre-denaturation at 95°C 5min, 95℃ for 30sec, 67℃ for 30sec, 72℃ for 1min, 30cycles, 72℃ for 10min, 4℃ for 10min; the PCR product was ligated to the pMD19-T vector, and the constructed vector was named T-COE. The porcine epidemic diarrhea virus S protein gene sequence SEQID NO.2 of the region; 1.3 Cloning of intestinal M-like cell targeting peptide L-lectin-β-GF gene Design a pair of primers for amplifying the L-lectin-β-GF gene with reference to the published L-lectin-β-GF gene sequence, the GenBank accession number of the L-lectin-β-GF gene sequence is BA000018.3, in The 5' end of the upstream primer introduces a 20 bp homology arm sequence with the 3' end of the porcine epidemic diarrhea virus S protein gene; the 5' end of the downstream primer introduces a 15 bp homology arm sequence with the 5' end of the pHT43 multi-cloning site. The XmaI restriction site was introduced between the two, wherein the underline is the restriction site, and the primer sequence is as follows: P5: ATAAAGATGATGATGATAAATTTRCGTCARCARCGACGAC, sequence number is SEQ ID NO.8; P6: CATTAGRCGGRCTRC CCCGGG AGTAAAATAATATGT, sequence number is SEQ ID NO.9; The genome extracted from Staphylococcus aureus strains was used as a template for PCR amplification; the PCR reaction system was 50 μL, and the PCR reaction conditions were: 95°C pre-denaturation for 5min, 95°C for 30sec, 69°C for 30sec, 72°C for 2min, 30cycles, 72°C 10min at 4°C for 10min; the PCR product was ligated to the pMD19-T vector with T4 ligase, and the constructed vectors were named T-L respectively. After sequencing, the L-lectin-β-GF gene sequence SEQ ID NO.3 was obtained; 1.4 Construction of recombinant expression vector pHT43-RCL Take SEQ ID NO.1 sequence, SEQ ID NO.2 sequence and SEQ ID NO.3 sequence as templates, use primers P1, P4 to carry out overlapping PCR amplification, the PCR reaction system is 50 μL, PCR reaction conditions: 95 ℃ pre-denaturation 5min , 95℃ for 30sec, 72℃ for 30sec, 72℃ for 4min, 30cycles, 72℃ for 10min, 4℃ for 10min; the PCR product was recovered and cloned into the pHT43 vector using the One Step Cloning Kit site-directed cloning kit, and the expression vector pHT43 was obtained after sequencing -RCL. 2. A recombinant Bacillus subtilis WB800N strain containing the recombinant expression plasmid pHT43-RCL of the intestinal M-like cell targeting peptide L-lectin-β-GF and PEDV S protein as claimed in claim 1. 3. the application of a recombinant Bacillus subtilis WB800N bacterial strain as claimed in claim 2, is characterized in that, described application is the biological application that stimulates mouse body to produce more effective PEDV specific immune response level, described application The recombinant Bacillus subtilis WB800N strain and the control recombinant Bacillus subtilis WB800N strain were used for oral immunization test of 6-week-old mice. The animal test and immune detection methods are as follows: Normal Bacillus subtilis WB800N strain without foreign gene in genetic material, control recombinant Bacillus subtilis WB800N strain and recombinant Bacillus subtilis WB800N expressing intestinal M-like cell targeting peptide L-lectin-β-GF and PEDV S protein The strains were orally immunized to 6-week-old mice with an oral dose of 10 ¹⁰ cfu/kg. The second and third immunizations were performed at the same dose after the first immunization for 7 days and 14 days. Serum and intestinal rinses were collected every week. The feeding cycle For 35 days; the indirect ELISA method was used to detect the changes of PEDV-specific IgG and specific SIgA levels. The detection method was as follows: PEDV antigen was coated overnight, 1% bovine serum albumin BSA was blocked at 37°C for 2 hours, and after washing, the sample to be tested was added 37 ℃ for 1 h, wash and add appropriately diluted horseradish peroxidase-labeled rabbit anti-mouse IgG or IgA antibody, 37 ℃ for 1 h, TMB chromogenic solution containing tetramethylbenzidine and H ₂ O ₂ after color development, enzyme labeling The OD ₄₅₀ was detected by the instrument; the results showed that the recombinant Bacillus subtilis expressing the intestinal M cell targeting peptide L-lectin-β-GF and PEDV S protein was more effective in stimulating the production of mice than the control recombinant Bacillus subtilis WB800N strain. level of PEDV-specific immune response.

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