CN111825774A

CN111825774A - Bivalent LTB toxin of porcine pathogenic escherichia coli as well as preparation process and application thereof

Info

Publication number: CN111825774A
Application number: CN202010957914.2A
Authority: CN
Inventors: 张渊魁; 唐青海
Original assignee: Zhaofenghua Biotechnology Nanjing Co Ltd
Current assignee: Zhaofenghua Biotechnology Nanjing Co Ltd
Priority date: 2020-09-14
Filing date: 2020-09-14
Publication date: 2020-10-27
Anticipated expiration: 2040-09-14
Also published as: CN111825774B

Abstract

The invention relates to the technical field of biological vaccines, in particular to a porcine pathogenic escherichia coli bivalent LTB toxin, a preparation process and an application thereof, wherein the porcine pathogenic escherichia coli bivalent LTB toxin gene has a nucleic acid sequence shown as SEQ ID No: 1 is shown. The invention also prepares a specific antibody, and the specific antibody and the swine fever spleen and lymph node vaccine are combined to prepare a bivalent vaccine, which can obviously enhance the level of the swine fever specific antibody and can simultaneously reduce the diarrhea rate of suckling piglets; the method has the advantages of achieving 'one-needle two-prevention', being more efficient, more economical and safer, effectively reducing the application of antibiotics in the aspect of treatment and reducing a series of problems caused by the excessive use of antibiotics.

Description

Bivalent LTB toxin of porcine pathogenic escherichia coli as well as preparation process and application thereof

Technical Field

The invention relates to the field of biotechnology, in particular to bivalent LTB toxin of pathogenic escherichia coli of a pig and application thereof.

Background

Escherichia coli is a small bacillus with blunt ends, has flagella around the body, can move, and belongs to gram-negative bacteria. Its antigenic components are complex, including: o antigen (thalli antigen), H antigen (flagellar antigen), and K antigen (capsular antigen). The Escherichia coli K antigen 103, O antigen 171 and H antigen 56 have been determined so far. The more ciliated serotypes there are, the less the absolute amount of each cilium should grow, and the less antigenic it will exhibit, without cross-reactivity between serotypes of the cilium antigen of enterotoxic E.coli.

Diarrheagenic Escherichia coli is a generic term for pathogenic Escherichia coli causing diarrhea in humans and animals, and mainly includes enteropathogenic Escherichia coli (EPEC), enterohemorrhagic Escherichia coli (EHEC), enteroinvasive Escherichia coli (EIEG), and enterotoxigenic Escherichia coli (ETEC). Enterotoxigenic escherichia coli (ETEC) is one of the main pathogenic bacteria causing infantile diarrhea and is also the pathogenic bacteria causing diarrhea of travelers. It is characterized by the ability to produce two types of virulence factors: adhesins that promote the binding and colonization of intestinal epithelial cells and enterotoxins responsible for the secretion of fluids, among which 2 toxic proteins secreted by ETEC: high molecular weight heat-Labile Toxin (LT) and low molecular weight heat-Stable Toxin (ST).

The high molecular weight calorimetric Labile Toxin (LT) is composed of 1A subunit (A subenit of Escherichia coli-lipid enterotox, LTA) and 5B subunit monomers (B subenit of Escherichia coli-lipid enterotox, LTB). The a subunit binds to GM1 gangliosides and glycoprotein receptors on cell membranes. The B subunit is the immunogenicity and receptor binding site of LT, is mainly specifically combined with GM1 ganglioside on the cell membrane of mammal to form a channel, and leads the A subunit to enter target cells, has mucosal adjuvant activity, and is known to be an effective mucosal immunoadjuvant. Because LTB removes a toxic A subunit of LT, namely receptor ganglioside GM1 which mainly recognizes intestinal mucosal epithelial cell membrane and is combined with the intestinal mucosal epithelial cell membrane to form a compound, the LTB has no toxicity, can be used as an adjuvant to assist foreign antigens to generate high-efficiency mucosal immunity on organisms, and is the key content in vaccine research. After LTB is used as a subcutaneous immune adjuvant to immunize organisms, the content of specific Ig G and sIg A which are produced by stimulation is higher, the mucosal immune response can be induced, and allergy can not be caused, because the Th1 pathway is activated. The enterotoxin plasmid and the drug-resistant plasmid of the escherichia coli can be transferred to other intestinal bacteria through combination and pairing, and even transferred to other non-escherichia coli intestinal bacteria to enable the intestinal bacteria to become pathogenic bacteria capable of producing enterotoxin and having drug resistance. In addition, the antigen and the antigen are combined to perform mucosal immunity, so that the antigen has higher immunogenicity, and can effectively induce and enhance the mucosal immune response to the enteroassociated virus. At present, there are few cases of research on fusion expression of different genotypes of pig LTB at home and abroad.

Classical swine fever (classic swine fever) is a highly-contagious and hemorrhagic infectious disease of pigs caused by Classical Swine Fever Viruses (CSFV), has wide epidemic range and high lethality, is one of the major threats of the pig industry, is determined as an infectious disease by China, and is listed as a class A virulent infectious disease by the world animal health Organization (OIE). The hog cholera lapinized attenuated vaccine successfully developed in the 50 th of the 20 th century in China has good immunogenicity, and for decades, the prevention and control measures of immunization with the hog cholera lapinized attenuated vaccine are adopted in China all the time, so that the hog cholera epidemic situation in China is effectively controlled, and the vaccine makes an important contribution to the prevention and control of the hog cholera all over the world.

However, in recent years, swine fever has a new epidemic in China: the condition of regional pandemics is rare, and the condition of sporadic occurrence is more frequent; the classical swine fever cases are rare but not common, and the problems of persistent infection and recessive infection are more serious. In clinical observation, infection of certain viruses can interfere the immune effect of the swine fever vaccine, so that the improvement of the vaccine quality and the conventional immunization work are very important for the prevention and control of the swine fever.

At present, the swine fever vaccines at home and abroad are prepared by single vaccine and are immunized by the single vaccine. Sows often carry out either prime or follow-up immunization. Because the suckling piglet is small in day age and difficult to start immune response quickly, the piglet mainly sucks colostrum to obtain immunity to infectious diseases such as swine fever. Therefore, the immune effect of the sow is directly related to the health and safety of the piglets. In recent years, the diarrhea caused by escherichia coli of piglets is becoming more serious, and the treatment effect of antibiotics is becoming worse with the increasing drug resistance of escherichia coli. China clearly defines that antibiotics are prohibited to be added into the feed in 7 months in 2020, and a new subject and challenge are provided for preventing and treating colibacillary diarrhea of piglets.

Disclosure of Invention

Based on the technical problems, the invention provides the bivalent LTB toxin of the porcine pathogenic escherichia coli, the specific antibody of the bivalent LTB toxin is prepared, the bivalent LTB toxin and the hog cholera spleen and lymph are combined to prepare a bivalent vaccine for immunizing a sow, and the bivalent LTB toxin can simultaneously prevent and treat the piglet hog cholera and enterotoxigenic porcine pathogenic escherichia coli diseases.

In order to achieve the purpose, the invention provides the following technical scheme:

transformation and multi-epitope fusion design of LTB gene

The invention fuses the I type and the II type, simultaneously expresses toxins of the two genotypes in one expression system through epitope optimization, and prepares a corresponding antibody for preventing and treating colibacillosis of piglets. The nucleotide sequence of the optimized bivalent LTB fusion toxin Open Reading Frame (ORF) is shown as SEQ ID No.1, and the amino acid sequence of the gene code is shown as SEQ No. 2. Which includes 5 core epitopes: epitope 1: NIQKLIALLFIVLN, epitope 2: YLCNQMRKIAMAAVL, epitope 3: VWGLALASEFDPRVPSS, epitope 4: ALIYPLYAHGAPQT, epitope 5: CVWNNKTPNSLPPFSVED are provided.

2. Prokaryotic expression of bivalent LTB toxin

(1) Connecting a bivalent LTB fusion toxin Open Reading Frame (ORF) with a pGEX4T-1 vector, constructing a recombinant expression vector pGEX-LTB, transforming BL21(DE3) competence, and screening to obtain a recombinant expression strain BL21(DE3) -pGEX-LTB;

(2) culturing recombinant expression strain BL21(DE3) -pGEX-LTB in LB culture medium, and inducing with IPTG at 37 deg.C and final concentration of 0.5mM for 4 h;

(3) centrifuging at 12000 r/min at 4 deg.C for 5min, and collecting thallus;

(4) adding 40mL of PBS (phosphate buffer solution) into each 1L of bacterial liquid, and fully suspending the bacterial;

(5) and (2) ultrasonically crushing the bacterial liquid under the conditions: the energy value is 60%, the ultrasonic treatment is carried out for 3s every 3s, and the total time is 0.5 h;

(6) centrifuging at 12000 r/min at 4 deg.C for 15min, and collecting precipitated protein;

(7) adding 30mL of PBS buffer solution into the precipitated protein;

(7) ultrasonic cracking: the energy value is 60%, the ultrasound is carried out for 5s every 5s, and the total time is 1 h;

(8) centrifuging at 12000 r/min at 4 deg.C for 15min, and collecting precipitated protein;

(9) and adding 10mL of PBS buffer solution into the precipitated protein, and performing vortex oscillation to prepare a purified LTB recombinant protein suspension.

3. Preparation of bivalent LTB antibody:

(1) mixing the LTB recombinant protein and a vaccine adjuvant according to a volume ratio of 1:1, wherein the mixing sequence is as follows: slowly adding the protein solution into the adjuvant solution until the final concentration of the protein is 0.1mg/mL, and simultaneously stirring and emulsifying at the rotating speed of 14000r/min for 0.5 h. The vaccine adjuvant is one or more of ISA201, ISA71VG, PET01 aluminum hydroxide and mineral oil.

(2) Immunizing the laying hens: the layer chicken is injected into the breast muscle, and the dosage is 1 mL/egg. 2 nd and 3 rd booster immunizations were performed on days 28 and 60 after the primary immunization, respectively. And (3) extracting eggs 7-30 days after 3 rd immunization, detecting the LTB specific yolk antibody titer by using a western blot method, and extracting and collecting the yolk antibody from the eggs by using PEG6000 when the antibody titer reaches more than 32000 times.

4. Bivalent LTB toxin and swine fever spleen and lymph node vaccine

Uniformly mixing 2-10 mu g of bivalent LTB toxin with 1 head (150 RID) of spleen stranguria vaccine, and freeze-drying.

When in use, the immunization is carried out by intramuscular injection, the immunization dose is 1-5 parts per time, and the secondary immunization is carried out at intervals of 28 days.

Compared with the prior art, the invention has the beneficial effects that:

(1) the invention designs two LTB toxins with different genotypes, and has wider application range.

(2) The preparation process of the bivalent LTB toxin is relatively simple, the preparation cost is low, and the obtained protein has good immunogenicity and reactogenicity;

(3) the toxin-specific antibody prepared by the invention has obvious prevention and treatment effect on enterotoxigenic escherichia coli (ETEC), is safe, efficient and residue-free, and can effectively reduce the dosage of antibiotics and reduce the harm brought by the use of antibiotics.

(4) The bivalent toxin can obviously enhance the level of the swine fever specific antibody, can simultaneously reduce the diarrhea rate of suckling piglets, can obviously improve the vaccine effect and quality of the swine fever spleen and stomach vaccine, and expands the functions of the vaccine.

(5) The vaccine is combined with swine fever and spleen stranguria vaccine to prepare a bivalent vaccine for immunizing sows, can simultaneously prevent and treat piglet swine fever and enterotoxigenic porcine pathogenic escherichia coli diseases, and can be used as a one-injection two-prevention method, so that the vaccine is more efficient, more economical and safer, the application of antibiotics in treatment is effectively reduced, and a series of problems caused by excessive use of antibiotics are reduced.

Drawings

FIG. 1 shows the fusion expression design of LTB toxin gene type I and type II.

FIG. 2 shows the restriction enzyme identification of Rosetta-pGEX.4T.1-LTB; wherein, M: DL 5000 DNA Marker; 1# -5 #: the restriction enzyme digestion product of Rosetta-pGEX.4T.1-LTB 1# -5 #.

FIG. 3 shows the restriction enzyme identification of recombinant expression vector BL21(DE3) -pGEX.4T-LTB. Wherein, M: DL 5000 DNASMarker; 1: BL21(DE3) -pGEX.4T-LTB.

FIG. 4 is a 0.5h inclusion body protein SDS-PAGE assay; wherein, M: a protein Marker; 1: BL21(DE3) -pGEX.4T-LTB induction; 2: BL21(DE3) -pGEX.4T-LTB was not induced; 3: BL21(DE3) -pGEx.4T induction; 4: BL21(DE3) -pGEX.4T was not induced; 5: BL21(DE3) induction; 6: BL21(DE3) was not induced.

FIG. 5 is a SDS-PAGE assay of 0.5h supernatant; wherein, M: a protein Marker; 1: BL21(DE3) -pGEX.4T-LTB induction; 2: BL21(DE3) -pGEX.4T-LTB was not induced; 3: BL21(DE3) -pGEx.4T induction; 4: BL21(DE3) -pGEX.4T was not induced; 5: BL21(DE3) induction; 6: BL21(DE3) was not induced.

FIG. 6 shows Western blotting identification of inclusion body protein samples at 0.5 h; wherein, M: a protein Marker; 1: BL21(DE3) -pGEX.4T-LTB induction; 2: BL21(DE3) -pGEX.4T-LTB was not induced; 3: BL21(DE3) -pGEX.4T induction; 4: BL21(DE3) -pGEX.4T was not induced; 5: BL21(DE3) induction; 6: BL21(DE3) was not induced.

FIG. 7 is 1h inclusion body protein SDS-PAGE detection; wherein, 1: BL21(DE3) -pGEX.4T-LTB induction; 2: BL21(DE3) -pGEX.4T-LTB was not induced; 3: BL21(DE3) -pGEx.4T induction; 4: BL21(DE3) -pGEX.4T was not induced; 5: BL21(DE3) induction; 6: BL21(DE3) was not induced.

FIG. 8 is a 1h supernatant SDS-PAGE assay; wherein, 1: BL21(DE3) -pGEX.4T-LTB induction; 2: BL21(DE3) -pGEX.4T-LTB was not induced; 3: BL21(DE3) -pGEx.4T induction; 4: BL21(DE3) -pGEX.4T was not induced; 5: BL21(DE3) induction; 6: BL21(DE3) was not induced.

FIG. 9 shows Western blotting identification of inclusion body protein samples for 1 h; wherein, 1: BL21(DE3) -pGEX.4T-LTB induction; 2: BL21(DE3) -pGEX.4T-LTB was not induced; 3: BL21(DE3) -pGEx.4T induction; 4: BL21(DE3) -pGEX.4T was not induced; 5: BL21(DE3) induction; 6: BL21(DE3) was not induced.

FIG. 10 is 2h inclusion body protein SDS-PAGE detection; wherein, 1: BL21(DE3) -pGEX.4T-LTB induction; 2: BL21(DE3) -pGEX.4T-LTB was not induced; 3: BL21(DE3) -pGEx.4T induction; 4: BL21(DE3) -pGEX.4T was not induced; 5: BL21(DE3) induction; 6: BL21(DE3) was not induced.

FIG. 11 is a 2h supernatant SDS-PAGE assay; wherein, 1: BL21(DE3) -pGEX.4T-LTB induction; 2: BL21(DE3) -pGEX.4T-LTB was not induced; 3: BL21(DE3) -pGEx.4T induction; 4: BL21(DE3) -pGEX.4T was not induced; 5: BL21(DE3) induction; 6: BL21(DE3) was not induced.

FIG. 12 shows Western blotting identification of inclusion body protein samples for 2 h; wherein, 1: BL21(DE3) -pGEX.4T-LTB induction; 2: BL21(DE3) -pGEX.4T-LTB was not induced; 3: BL21(DE3) -pGEx.4T induction; 4: BL21(DE3) -pGEX.4T was not induced; 5: BL21(DE3) induction; 6: BL21(DE3) was not induced.

FIG. 13 shows 4h inclusion body protein SDS-PAGE detection; wherein, 1: BL21(DE3) -pGEX.4T-LTB induction; 2: BL21(DE3) -pGEX.4T-LTB was not induced; 3: BL21(DE3) -pGEx.4T induction; 4: BL21(DE3) -pGEX.4T was not induced; 5: BL21(DE3) induction; 6: BL21(DE3) was not induced.

FIG. 14 is a 4h supernatant SDS-PAGE assay; wherein, 1: BL21(DE3) -pGEX.4T-LTB induction; 2: BL21(DE3) -pGEX.4T-LTB was not induced; 3: BL21(DE3) -pGEx.4T induction; 4: BL21(DE3) -pGEX.4T was not induced; 5: BL21(DE3) induction; 6: BL21(DE3) was not induced.

FIG. 15 shows Western blotting identification of inclusion body protein samples for 4 h; wherein, M: a protein Marker; 1: BL21(DE3) -pGEX.4T-LTB induction; 2: BL21(DE3) -pGEX.4T-LTB was not induced; 3: BL21(DE3) -pGEx.4T induction; 4: BL21(DE3) -pGEX.4T was not induced; 5: BL21(DE3) induction; 6: BL21(DE3) was not induced.

FIG. 16 shows SDS-PAGE detection of supernatant protein samples at different induction concentrations; wherein, M: a protein Marker; 1: BL21(DE3) -pGEX.4T-LTB (1 mmol/L); 2: BL21(DE3) -pGEX.4T-LTB (0.5 mmol/L); 3: BL21(DE3) -pGEX.4T-LTB (0.25 mmol/L); 4: BL21(DE3) -pGEX.4T-LTB (0.1 mmol/L); 5: BL21(DE3) -pGEX.4T-LTB (0.05 mmol/L); 6: BL21(DE3) -pGEX.4T-LTB (0.025 mmol/L); 7: BL21(DE3) -pGEX.4T-LTB was not induced; 8: BL21(DE3) -pGEX.4T was not induced; 9: BL21(DE3) was not induced.

FIG. 17 shows SDS-PAGE detection of inclusion body protein samples at different induction concentrations; wherein, M: a protein Marker; 1: BL21(DE3) -pGEX.4T-LTB (1 mmol/L); 2: BL21(DE3) -pGEX.4T-LTB (0.5 mmol/L); 3: BL21(DE3) -pGEX.4T-LTB (0.25 mmol/L); 4: BL21(DE3) -pGEX.4T-LTB (0.1 mmol/L); 5: BL21(DE3) -pGEX.4T-LTB (0.05 mmol/L); 6: BL21(DE3) -pGEX.4T-LTB (0.025 mmol/L); 7: BL21(DE3) -pGEX.4T-LTB was not induced; 8: BL21(DE3) -pGEX.4T was not induced; 9: BL21(DE3) was not induced.

FIG. 18 shows Western blotting identification of inclusion body protein samples at different induced concentrations; wherein, M: a protein Marker; 1: BL21(DE3) -pGEX.4T-LTB (1 mmol/L); 2: BL21(DE3) -pGEX.4T-LTB (0.5 mmol/L); 3: BL21(DE3) -pGEX.4T-LTB (0.25 mmol/L); 4: BL21(DE3) -pGEX.4T-LTB (0.1 mmol/L); 5: BL21(DE3) -pGEX.4T-LTB (0.05 mmol/L); 6: BL21(DE3) -pGEX.4T-LTB (0.025 mmol/L); 7: BL21(DE3) -pGEX.4T-LTB was not induced; 8: BL21(DE3) -pGEX.4T was not induced; 9: BL21(DE3) was not induced.

FIG. 19 shows SDS-PAGE detection of supernatant protein samples at different induction temperatures; wherein, M: a protein Marker; 1: BL21(DE3) -pGEX.4T-LTB (37 ℃); 2: BL21(DE3) -pGEX.4T-LTB (30 ℃); 3: BL21(DE3) -pGEx.4T-LTB (25 ℃ C.); 4: BL21(DE3) -pGEX.4T-LTB (20 ℃ C.); 5: BL21(DE3) -pGEX.4T-LTB (15 ℃ C.); 6: BL21(DE3) -pGEX.4T-LTB (10 ℃ C.).

FIG. 20 shows SDS-PAGE detection of inclusion body protein samples at different induction temperatures; wherein, M: a protein Marker; 1: BL21(DE3) -pGEX.4T-LTB (37 ℃); 2: BL21(DE3) -pGEX.4T-LTB (30 ℃); 3: BL21(DE3) -pGEx.4T-LTB (25 ℃ C.); 4: BL21(DE3) -pGEX.4T-LTB (20 ℃ C.); 5: BL21(DE3) -pGEX.4T-LTB (15 ℃ C.); 6: BL21(DE3) -pGEX.4T-LTB (10 ℃ C.).

FIG. 21 shows Western blotting identification of supernatant protein samples at different induction temperatures; wherein, M: a protein Marker; 1: BL21(DE3) -pGEX.4T-LTB (37 ℃); 2: BL21(DE3) -pGEX.4T-LTB (30 ℃); 3: BL21(DE3) -pGEx.4T-LTB (25 ℃ C.); 4: BL21(DE3) -pGEX.4T-LTB (20 ℃ C.); 5: BL21(DE3) -pGEX.4T-LTB (15 ℃ C.); 6: BL21(DE3) -pGEX.4T-LTB (10 ℃ C.).

FIG. 22 shows Western blotting identification of inclusion body protein samples at different induction temperatures; wherein, M: a protein Marker; 1: BL21(DE3) -pGEX.4T-LTB (37 ℃); 2: BL21(DE3) -pGEX.4T-LTB (30 ℃); 3: BL21(DE3) -pGEx.4T-LTB (25 ℃ C.); 4: BL21(DE3) -pGEX.4T-LTB (20 ℃ C.); 5: BL21(DE3) -pGEX.4T-LTB (15 ℃ C.); 6: BL21(DE3) -pGEX.4T-LTB (10 ℃ C.).

FIG. 23 shows Western blotting of supernatant and inclusion body protein samples; wherein, M: a protein Marker; 1: BL21(DE3) -pGEX.4T-LTB 42 ℃ supernatant; 2: BL21(DE3) -pGEX.4T-LTB 42 ℃ inclusion body; 3: BL21(DE3) -pGEx.4T-LTB supernatant at 37 ℃; 4: BL21(DE3) -pGEX.4T-LTB inclusion bodies at 37 ℃; 5: BL21(DE3) -pGEX.4T-LTB supernatant at 30 ℃; 6: BL21(DE3) -pGEX.4T-LTB inclusion bodies at 30 ℃; 7: BL21(DE3) -pGEX.4T-LTB supernatant at 10 ℃; 8: BL21(DE3) -pGEX.4T-LTB 10 ℃ Inclusion bodies.

FIG. 24 is a western blotting assay for yolk antibody titers.

FIG. 25 shows the effect of bivalent LTB toxin on the immune response of spleen-strangle vaccine of swine fever.

FIG. 26 shows the effect of bivalent LTB toxin and classical swine fever-spleen-lymph-vaccine combined vaccine on prevention of diarrhea in suckling piglets.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 transformation of LTB Gene and design of Multi-epitope fusion

LTB toxin can be divided into two genotypes of type I (366 bp) and type II (375 bp), and the length of the nucleotide fragment is the same. In the experiment, the type I and the type II are fused, and toxins of two genotypes are simultaneously expressed in an expression system through epitope optimization to prepare corresponding antibodies for preventing and treating colibacillosis of piglets. The specific fusion expression design scheme is shown in FIG. 1, and a connecting sequence is inserted between type I and type II.

The nucleotide sequence of the optimized fusion Open Reading Frame (ORF) is shown as SEQ ID No: 1 is shown.

The amino acid sequence coded by the nucleotide sequence of the optimized fusion Open Reading Frame (ORF) is shown as SEQ ID No: 2, respectively.

Which includes 5 core epitopes: epitope 1: NIQKLIALLFIVLN, epitope 2: YLCNQMRKIAMAAVL, epitope 3: VWGLALASEFDPRVPSS, epitope 4: ALIYPLYAHGAPQT, epitope 5: CVWNNKTPNSLPPFSVED are provided.

Example 2 prokaryotic expression of bivalent LTB toxin

(1) Construction of recombinant expression strains

And carrying out double enzyme digestion identification on the stored recombinant plasmid Rosetta-pGEX.4T.1-LTB 1# -5# by adopting two endonucleases of BamH I and Sal I.

The results show that the Rosetta-pGEX.4T.1-LTB is 3 bands, namely a carrier band with the molecular weight of 5000 bp, a band of a target gene with the molecular weight of 789 bp and a hybrid band with the molecular weight of about 4800 bp, which are suspected to be incomplete in enzyme digestion. The result shows that the recombinant plasmid Rosetta-pGEX-4T-LTB is positive. As shown in fig. 2.

The plasmid (1 #) of Rosetta-pGEX.4T.1-LTB with correct identification result is transformed into BL21(DE3) competent cells, after single clone bacteria are selected, amplification culture is carried out, a small plasmid extraction kit is used for extracting the plasmid, and the plasmid is subjected to double enzyme digestion identification by endonucleases BamH I and Xho I, the result shows that the recombinant strain BL21(DE3) -pGEX.4T.1-LTB is two bands, respectively a carrier band with the molecular weight of 5000 bp and a band of a target gene with the molecular weight of 789 bp, and the recombinant vector BL21(DE3) -pGEX.4T.1 is a positive vector and the corresponding strain BL21(DE3) -pGEX.4T.1-LTB is a positive strain. As shown in fig. 3.

(2) Inducible expression of recombinant proteins

A. Optimization of optimal induction time for recombinant protein prokaryotic expression

Dividing the test tube containing the activated bacteria liquid into two groups, wherein one group is added with the test tube with the final concentration of 1.0 mmol.L^-1The IPTG of (1) and the other group without adding the inducer IPTG as a control group. The set temperature is 37 ℃ and the rotating speed is 220 r.min^-1Then, recombinant strain BL21(DE3) -pGEX-4T-LTB is induced for 0.5h, 1h, 2h and 4h respectively, andsampling once at 0.5h, 1mL of bacteria per tube each time, immediately collecting the bacteria, extracting a protein sample by using a bacterial protein extraction solution, and storing the protein sample in a refrigerator at the temperature of-80 ℃ for later use.

As shown in FIGS. 4-15, the results of SDS-PAGE showed that BL21(DE3) -pGEX-4T-LTB inclusion body protein expression with a protein band at 54kDa when the induction time was 0.5h, whereas the non-induced control bacteria and the induced empty vector and empty bacteria have no specific bands, and no significant characteristic band was observed in the induction supernatant. Meanwhile, after 1h, 2h and 4h of induction, characteristic bands do not appear in the inclusion bodies and the supernatant. The preliminary result is judged that the induction time is short, the protein expression amount is slightly high, and the recombinant protein BL21(DE3) -pGEX-4T-LTB is only expressed in the form of inclusion body in the escherichia coli and has no soluble expression.

B. Optimization of recombinant protein prokaryotic expression optimal IPTG induction concentration

After the optimal induction time condition of BL21(DE3) -pGEX-4T-LTB recombinant protein is determined to be 0.5h, different concentrations of inducer IPTG are set to induce and express the protein, and the concentrations are respectively set to be 1 mmol.L^-1、0.5 mmol•L^-1、0.25 mmol•L^-1、0.1 mmol•L^-1、0.05 mmol•L^-1And 0.025 mmol.L^-1These six different concentrations of experimental groups. Meanwhile, empty carriers and empty bacteria are set as a control group and an uninduced group are mutually controlled, as shown in figures 16-18, SDS-PAGE detection and Western blotting identification show that when the concentration of an inducer IPTG is 0.1 mmol.L^-1When the recombinant protein is used, BL21(DE3) -pGEX-4T-LTB inclusion body protein expression level is the highest, BL21(DE3) -pGEX-4T-LTB recombinant protein is only expressed in the form of inclusion body, target protein is not found in supernatant, the protein can be expressed only after the induction of inducer IPTG, and the non-induced bacterial liquid is not expressed.

C. Optimization of optimal induction temperature condition for recombinant protein prokaryotic expression

After the optimal induction time condition and IPTG concentration are obtained, the experiment is optimized again, and different induction temperatures are adopted: protein expression is carried out at six temperatures of 37 ℃, 30 ℃, 25 ℃, 20 ℃, 15 ℃ and 10 ℃.

As shown in FIGS. 19-23, SDS-PAGE showed that BL21(DE3) -pGEX-4T-LTB inclusion body protein was expressed in the largest amount at 37 ℃ and a part of the inclusion body protein was expressed at 30 ℃ and that the inclusion body protein was expressed in a smaller amount at 25 ℃ compared to 37 ℃ and 30 ℃ and that LTB inclusion body protein was degraded at 20 ℃ and other experimental temperatures. When the temperature is 10 ℃, a suspected target protein band appears near the molecular weight of the target protein, the expression amount is large, and after Western blotting identification, the protein is not the target protein. After experimental exploration is carried out on the temperatures, it is preliminarily considered that the higher the temperature is, the larger the expression amount of BL21(DE3) -pGEX-4T-LTB recombinant protein is, therefore, a group of experimental groups is additionally established, bacterial liquid with the same culture conditions is added with an inducer IPTG, then the bacterial liquid is placed in an environment of 42 ℃ for induction expression, after induction is finished, the bacterial bodies are collected according to the same method, protein is extracted, a protein sample is prepared, and after Western blotting identification, the result shows that BL21(DE3) -pGEX-4T-LTB recombinant protein can still be expressed at 42 ℃, but the expression amount is smaller than that at 37 ℃, the protein is easy to degrade at high temperature, and the difficulty is increased for extraction and storage of the protein, so the optimal expression temperature is 37 ℃.

The recombinant protein BL21(DE3) -pGEX.4T-LTB is mainly expressed in the form of inclusion body, has the molecular weight of about 54KDa, exists in the form of inclusion body, and has the final concentration of 0.1 mmol.L at 37 DEG C^-1The expression quantity of the inclusion body reaches the highest under the condition of IPTG induction for 0.5 h.

EXAMPLE 3 preparation of bivalent antibodies of type I and type II LTB toxins from porcine pathogenic E.coli

Purifying the protein, mixing with an immunologic adjuvant (MONTANETMISA 201 VG), fully emulsifying to prepare immunogen, immunizing laying hens by adopting a breast intramuscular injection mode, wherein the amount of the protein immunized by each chicken is 100-200 mug, and immunizing the 2 nd time and the 3 rd time by adopting the same immunogen in the same mode at 28d and 60 d after primary immunization respectively. Non-immunized chickens were set as negative controls. Collecting eggs 7-10 days after the three-stage immunization to extract the composite yolk antibody IgY, and measuring the titer of the antibody by Western blotting.

The yolk antibody extraction process comprises the following steps: adding 9 parts by volume of deionized water into 1 part of yolk liquid according to the volume ratio of 1:10, and uniformly stirring; adjusting the pH value of the yolk solution to 5.0-5.2 by using 1 mol/L HCl. Centrifuging at 4 deg.C at 10000 r/min for 25 min, collecting supernatant, adding 19% (W/V) sodium sulfate, stirring to dissolve, and standing at room temperature for 2 hr. Centrifuging at 10000 r/min at 4 deg.C for 25 min, removing supernatant, weighing precipitate, adding PBS (pH 7.4) to precipitate for dissolving (dissolving yolk stock solution per 10mL with 1mL PBS solution), and storing at 20 deg.C.

And collecting eggs 7d after the three-immunization to extract the composite yolk antibody IgY, and measuring the titer of the antibody by Western blotting to 8000X. As shown in fig. 24.

EXAMPLE 4 evaluation of effective prophylactic and therapeutic dose of porcine pathogenic E.coli LTB toxin type I and type II bivalent yolk antibody preparations

The yolk antibody preparation prepared by the method is extracted to develop animal experimental research of prevention and treatment effects. Balb/c mice were 30 mice weighing 25g and randomized into five groups: A. groups B and C, 10 per group; group C is untreated control group; group B is a challenge control group to which no antibody preparation was administered; a is a test group, and the first oral administration of the yolk antibody preparation mixture is carried out 2h before the challenge, and then the porcine pathogenic escherichia coli LTB toxin I and II bivalent yolk antibody preparation is orally administered once every 12h, 24 h and 48h, wherein each dosage is 0.2 mL. 4h after the second administration, each mouse in the A group and the B group orally takes ETEC E.Coli 108cfu/mL for attacking and infecting, and the C group is a non-infected healthy control and is separately fed. And continuously observing for 7 days, and counting the diarrhea rate and the death rate.

Through experimental observation statistics, it can be seen from table 1 that: group C had no diarrhea and no mortality. Group B experienced 100% diarrhea and mortality, and all died on day 4 after virus challenge. Group a had a diarrhea rate of 60% and a mortality rate of 30%. Therefore, the antibody preparation has better control effect. The fusion protein of the type I and the type II of the porcine pathogenic escherichia coli LTB toxin prepared by the invention has better immunogenicity, can generate higher low-degree antibody, can obviously reduce the diarrhea rate and the death rate caused by pathogenic escherichia coli infection by a high-titer egg yolk antibody preparation, and has better application prospect.

TABLE 1 effective control dose of pathogenic E.coli LTB toxin type I and type II bivalent antibody preparations

	Group A	Group B	Group C
				Number of diarrhea	6	10	0
Number of deaths	3	10	0
				Rate of diarrhea	60%	100%	0
Mortality rate	30%	100%	0

Example 5 Effect of bivalent LTB toxin on the Immunity of spleen and lymph of hog cholera

The spleen stranguria single vaccine is prepared according to the existing preparation procedure, 2-10 mu g of bivalent LTB toxin is uniformly mixed with 1 head (150 RID) of spleen stranguria single vaccine, and the mixture is freeze-dried to prepare the bivalent vaccine. 80 pigs, 80 days old, with negative antigen and antibody were selected and randomly divided into 6 groups: groups A-H, 10 heads each.

Group A is injected with 1 part of hog cholera spleen gonorrhea vaccine (150 RID) through muscle, group B is injected with 1 part of bivalent LTB toxin (1 mu g) and 1 part of spleen gonorrhea bivalent vaccine (containing the spleen gonorrhea 150 RID), group C is injected with 1 part of bivalent LTB toxin (2 mu g) and 1 part of spleen gonorrhea bivalent vaccine (containing the spleen gonorrhea 150 RID), and group D is injected with 1 part of bivalent LTB toxin (4 mu g) and 1 part of spleen gonorrhea bivalent vaccine (containing the spleen gonorrhea 150 RID). Group E was injected with 1 part of bivalent LTB toxin (6. mu.g) and 1 part of bigeminy vaccine containing splenic gonorrhea 150 RID. Group F was injected with 1 part of bivalent LTB toxin (8. mu.g) and 1 part of spleen-stranguria bivalent vaccine (containing 150 RID). Group G was injected with 1 part of bivalent LTB toxin (10. mu.g) and 1 part of bigeminy vaccine containing splenic gonorrhea toxin 150 RID. Group H was not immunized. Groups A to G are immunized twice, and the second immunization is carried out 21d after the first immunization. Collecting blood in the anterior vena cava 7, 14, 21, 28, 35, 42 and 57 days after the first immunization respectively, preparing serum, and measuring the CSFV antibody titer by adopting an immunoperoxidase monolayer cell staining method.

As can be seen from FIG. 25, the bivalent LTB toxin can significantly enhance the immune effect of the spleen lymph of swine fever, and has dose dependence (between 2 and 8 ug), with no difference between 10 ug and 8 ug.

Example 6 Effect of bivalent LTB toxin and classical swine fever-spleen-stranguria bivalent vaccine on prevention effect of diarrhea in suckling piglets

2-10 mu g of bivalent LTB toxin is uniformly mixed with 1 part (150 RID) of spleen stranguria vaccine, and the mixture is freeze-dried to prepare the bivalent vaccine. 30 sows pregnant for 80 days were selected, and randomized into 3 groups: group A, group B and group C, 10 of each group, 1 part of swine fever spleen strangury vaccine (150 RID) is injected into the group A through muscle, 1 part of bivalent LTB toxin (8 mu g) and 1 part of spleen strangury virus bivalent vaccine (containing the spleen strangury virus 150 RID) are injected into the group B through muscle, and the group C does not carry out immunization. Groups A and B were immunized twice, 21d after the first immunization. And (4) counting the diarrhea rate of each group of suckling piglets in the lactation period (21 days). As can be seen from FIG. 26, the diarrhea rate of group A is 21.2%, the diarrhea rate of group B is 7.5%, and the diarrhea rate of group C is 21.5, which are significantly reduced compared with the diarrhea rate of A, C and group B, indicating that the spleen stranguria vaccine is added with bivalent LTB toxin (8 μ g) for simultaneous immunization, which can effectively reduce the diarrhea rate of the suckling piglets and improve the production performance.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Sequence listing

<110> Mtoyowa Biotechnology (Nanjing) Ltd

<120> porcine pathogenic escherichia coli bivalent LTB toxin, preparation process and application

<160>2

<170>SIPOSequenceListing 1.0

<210>1

<211>783

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

atgaacattc aaaagctaat tgcgttattg tttattgtct taaatattgc atcactacca 60

acatatgctg gtgtaagtaa aacttttaag gataactgcg cttctactac ggcaaaactt 120

gtacagagtg ttcagttggt aaatatctcg tctgatgtaa ataaggacag taagggaatt 180

tatatatcaa gctcagcagg aaaaacatgg tttattccgg gggggcagta ttaccctgat 240

aactatctat gtaatcaaat gagaaaaata gcaatggctg cagttctttc taacgtaagg 300

gtaaatctat gtccgagtga agcatatact ccgaatcatg tatggggact tgcactggca 360

tcagaattcg atccccgggt accgagctcg atgaataaag taaaatgtta tgttttatgt 420

acggccttaa tataccctct atatgcacac ggagctcccc agactattac agaactatgt 480

tcggaatatc gcaacacaca aatatatacg ataaatgaca agatactatc atatacggaa 540

tcgatggcag gcaaaagaga aatggttatc attacattta agagcggcga aacatttcag 600

ctcgaagacc cgggcagtca acatatagac tcccaggcaa aagccattga aaggatgaag 660

gacacattaa gaatcacata tctgaccgag accagaattg ataaattatg tgtatggaat 720

aataaaaccc ccaattcact tccgccattc agtgtggaag accaccacca ccaccaccac 780

taa 783

<210>2

<211>260

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>2

Met Asn Ile Gln Lys Leu Ile Ala Leu Leu Phe Ile Val Leu Asn Ile

1 5 10 15

Ala Ser Leu Pro Thr Tyr Ala Gly Val Ser Lys Thr Phe Lys Asp Asn

20 25 30

Cys Ala Ser Thr Thr Ala Lys Leu Val Gln Ser Val Gln Leu Val Asn

35 40 45

Ile Ser Ser Asp Val Asn Lys Asp Ser Lys Gly Ile Tyr Ile Ser Ser

50 55 60

Ser Ala Gly Lys Thr Trp Phe Ile Pro Gly Gly Gln Tyr Tyr Pro Asp

65 70 75 80

Asn Tyr Leu Cys Asn Gln Met Arg Lys Ile Ala Met Ala Ala Val Leu

85 90 95

Ser Asn Val Arg Val Asn Leu Cys Pro Ser Glu Ala Tyr Thr Pro Asn

100 105 110

His Val Trp Gly Leu Ala Leu Ala Ser Glu Phe Asp Pro Arg Val Pro

115 120 125

Ser Ser Met Asn Lys Val Lys Cys Tyr Val Leu Cys Thr Ala Leu Ile

130 135 140

Tyr Pro Leu Tyr Ala His Gly Ala Pro Gln Thr Ile Thr Glu Leu Cys

145 150 155 160

Ser Glu Tyr Arg Asn Thr Gln Ile Tyr Thr Ile Asn Asp Lys Ile Leu

165 170 175

Ser Tyr Thr Glu Ser Met Ala Gly Lys Arg Glu Met Val Ile Ile Thr

180 185 190

Phe Lys SerGly Glu Thr Phe Gln Leu Glu Asp Pro Gly Ser Gln His

195 200 205

Ile Asp Ser Gln Ala Lys Ala Ile Glu Arg Met Lys Asp Thr Leu Arg

210 215 220

Ile Thr Tyr Leu Thr Glu Thr Arg Ile Asp Lys Leu Cys Val Trp Asn

225 230 235 240

Asn Lys Thr Pro Asn Ser Leu Pro Pro Phe Ser Val Glu Asp His His

245 250 255

His His His His

260

Claims

1. A swine pathogenic Escherichia coli bivalent LTB toxin is characterized in that: the nucleic acid sequence of the porcine pathogenic escherichia coli bivalent LTB toxin gene is shown as SEQ ID No: 1 is shown.

2. The swine pathogenic escherichia coli bivalent LTB toxin according to claim 1, wherein: the amino acid sequence of the gene code of the porcine pathogenic escherichia coli bivalent LTB toxin is shown as SEQ ID No: 2, respectively.

3. The swine pathogenic escherichia coli bivalent LTB toxin according to claim 2, wherein: comprises 5 core antigen epitopes which are respectively:

epitope 1: NIQKLIALLFIVLN the flow of the air in the air conditioner,

epitope 2: YLCNQMRKIAMAAVL the flow of the air in the air conditioner,

epitope 3: VWGLALASEFDPRVPSS the flow of the air in the air conditioner,

epitope 4: ALIYPLYAHGAPQT the flow of the air in the air conditioner,

epitope 5: CVWNNKTPNSLPPFSVED are provided.

4. A process for the preparation of the swine pathogenic escherichia coli bivalent LTB toxin according to any one of claims 1 to 3, characterized in that:

(1) connecting a bivalent LTB toxin open reading frame ORF with a pGEX4T-1 vector, constructing a recombinant expression vector pGEX-LTB, transforming BL21(DE3) competence, and screening to obtain a recombinant expression strain BL21(DE3) -pGEX-LTB;

(2) culturing recombinant expression strain BL21(DE3) -pGEX-LTB, LB, inducing at 37 deg.C with IPTG with final concentration of 0.5mM for 4 h;

(3) centrifuging at 12000 r/min at 4 deg.C for 5min, and collecting thallus;

(7) adding 30mL of PBS buffer solution into the precipitated protein;

5. A preparation process of a bivalent LTB antibody is characterized by comprising the following steps:

(1) mixing the LTB recombinant protein suspension prepared according to claim 4 with a vaccine adjuvant in a volume ratio of 1:1, in the order of: slowly adding the protein solution into the adjuvant solution until the final concentration of the protein is 0.1mg/mL, and stirring and emulsifying at the rotating speed of 14000r/min for 0.5 h;

(2) immunizing the laying hens: carrying out breast intramuscular injection on the laying hens, wherein the dosage is 1mL per laying hen; 2 nd and 3 rd booster immunizations were performed on days 28 and 60 after the primary immunization, respectively; and (3) extracting eggs 7-30 days after 3 rd immunization, detecting the LTB specific yolk antibody titer by using a western blot method, and extracting and collecting the yolk antibody from the eggs by using PEG6000 when the antibody titer reaches more than 32000 times.

6. The process for the preparation of a bivalent LTB antibody according to claim 5, wherein: the vaccine adjuvant is one or more of ISA201, ISA71VG, PET01 aluminum hydroxide and mineral oil.

7. Use of the swine pathogenic escherichia coli bivalent LTB toxin according to any one of claims 1 to 3, wherein: and the vaccine is prepared into a bivalent vaccine together with the swine fever spleen and lymph node vaccine.

8. The use of the bivalent LTB toxin of pathogenic escherichia coli of claim 7, wherein: uniformly mixing 2-10 mu g of bivalent LTB toxin with 1 head of 150RID of spleen stranguria vaccine, and freeze-drying.