CN101560247B - Mucous membrane immunologic adjuvant using heat-sensitive colitoxin dual-mutant as vaccine - Google Patents

Abstract

The invention discloses a separated heat-sensitive colitoxin mutant protein. Compared with A subunit of wild type heat-sensitive colitoxin, A subunit of the protein has the 63th position of the amino acid sequence being K, the 192nd position being G, or the 72nd position being R, and the 192nd position being G. The invention also discloses a coding gene of the protein, a carrier or cell containingthe gene, an adjuvant composite containing the protein, and a production method of the protein.

Description

Escherichia coli heat-sensitive enterotoxin double mutant as an adjuvant for vaccine mucosal immunity

technical field

The invention belongs to the field of biotechnology, and more specifically, the invention relates to a heat-sensitive enterotoxin double mutant of Escherichia coli, its coding gene, a vector or cell containing the gene, an adjuvant composition containing the protein, and a production method of the protein .

Background technique

Mucosal immunity is an important part of the body's immune defense system. Immunoprophylaxis through the mucosa can not only induce the production of immune antibodies in the mucosa, but also stimulate a systemic immune response. Therefore, mucosal immunization is the best way to prevent respiratory and digestive system diseases. The best way to immunize. However, because many vaccines induce low levels of antibodies during mucosal vaccination, they cannot produce sufficient immune protection. Therefore, appropriate mucosal adjuvants are required to induce higher levels of mucosal IgA antibodies. There are many kinds of mucosal immune adjuvants currently being studied, among which bacterial metabolites, heat-sensitive enterotoxin and cholera toxin as vaccine mucosal immune adjuvants are of particular concern.

Heat-labile enterotoxin (LT) produced by Enterotoxic Escherichia coli (ETEC) is one of the toxins that cause diarrhea in humans and mammals. It consists of 1 A subunit and 5 B subunits. The B subunit can mainly regulate the combination of LT and GM1 on the cell membrane, and the A subunit has ADP-ribosyltransferase activity and is the main virulence factor. The complete LT A subunit can be cleaved by trypsin into A1 and A2, A2 is located at the C-terminus of A1, and the two are connected by a disulfide bond, in which A1 is the toxic site, and A2 is connected to the LT B subunit. After entering the target cells, the disulfide bonds are reduced and LT exhibits toxicity.

Many studies have shown that LT is a potential mucosal immune adjuvant, but its clinical application is limited due to its strong toxicity. At present, people are trying to achieve the purpose of reducing the toxicity of LT while retaining its adjuvant function through site-directed mutagenesis technology. Studies have found that the sites related to the toxicity of LT are mainly concentrated on the A subunit, and the purpose of reducing the toxicity of LT can be achieved by changing the amino acid sites. Mutations at many sites will affect its ADP-ribosyltransferase activity, and the more common mutants at sites 7, 44, 49, 50, 63, 72, 110, 112, and 146 show Different degrees of residual toxicity and different degrees of adjuvant activity led to mixed conclusions.

The research on LT mutants in the prior art mainly focuses on the construction of single-site mutants, and the toxicity of single-site mutants may not be completely lost, and the probability of regaining virulence due to back mutation is relatively high. Theoretically, the possibility of reverse mutation will be reduced by constructing multi-site LT mutants. However, whether multi-site mutations will affect the activity of LT as a mucosal immune adjuvant is still unknown, and it is still necessary to find a suitable multi-site mutant. A combination of mutation positions was used to enable LT to exert as strong an immune adjuvant activity as possible while minimizing toxicity.

Contents of the invention

The object of the present invention is to provide an Escherichia coli heat-sensitive enterotoxin double mutant with low toxicity and excellent immune adjuvant activity, its coding gene, a vector or cell containing the gene, an adjuvant composition containing the protein, and the protein production method.

In the first aspect of the present invention, there is provided an isolated Escherichia coli heat-sensitive enterotoxin mutant protein, said protein comprising the A subunit and the B subunit of the Escherichia coli heat-sensitive enterotoxin protein, and the amino acid sequence of the A subunit Relative to the amino acid sequence of the A subunit of wild-type Escherichia coli heat-sensitive enterotoxin,

Its 63rd position is K, and its 192nd position is G;

its 72nd position is R and its 192nd position is G; or

Its 63rd position is K, its 72nd position is R, and its 192nd position is G.

In another preferred example, the protein contains 1 A subunit of Escherichia coli heat-sensitive enterotoxin protein and 5 B subunits of Escherichia coli heat-sensitive enterotoxin protein.

In another preferred example, the protein is basically composed of Escherichia coli heat-sensitive enterotoxin A subunit and B subunit.

In another preferred example, the protein is basically composed of 1 A subunit and 5 B subunits.

In a second aspect of the present invention, an isolated nucleic acid encoding said protein is provided.

In the third aspect of the present invention, there is provided a vector containing the nucleic acid.

In the fourth aspect of the present invention, there is provided a cell containing the vector, or the nucleic acid integrated in its genome.

In the fifth aspect of the present invention, there is provided a method for producing the protein, comprising: culturing the cells under conditions suitable for expression, so as to express the protein.

In another preferred example, the method includes:

(1) expressing the A subunit and the B subunit of the heat-sensitive enterotoxin mutant protein of Escherichia coli in the cell;

(2) Collect the cells of (1), resuspend the cells with a denaturant, and break the cells;

(3) Mix the cell disruption product of (2), and collect the supernatant by centrifugation;

(4) Separate the protein containing A subunit and B subunit from the supernatant obtained in (3), and refold to obtain the desired protein.

In another preferred example, the denaturant is NTAO.

In another preferred example, the NTAO contains: Tris-HCl, NaCl, and glycerol.

In another preferred example, the formula of the NTAO is: 20mmol/L Tris-HCl, 0.5mol/LNaCl, 10% glycerin.

In another preferred example, the cells are Escherichia coli cells.

In the sixth aspect of the present invention, the use of the protein is provided for preparing an adjuvant for mucosal immunity.

In the seventh aspect of the present invention, there is provided a composition for mucosal immunization, which contains: an effective amount of the protein as an adjuvant for mucosal immunization.

In another preferred embodiment, the effective amount of said protein is 0.0001-20wt% (more preferably 0.001-10wt%).

In another preferred example, the composition further contains: an effective amount (such as 0.0001-20wt%; more preferably 0.001-10wt%) of a vaccine for mucosal immunity.

Description of drawings

Figure 1 shows the agarose gel electrophoresis analysis of pET30a-LTK63/G192.

Among them, 1: LTK63/G192I; 2: LTK63/G192II; 3: PCR amplification of LTK63/G192 gene; 4: DL 2000; 5: double enzyme digestion identification of recombinant plasmid; 6: PCR identification of pET30a-LTK63/G192 recombinant plasmid.

Figure 2 shows the agarose gel electrophoresis analysis of pET30a-LTR72/G192.

Among them, 1: LTR72/G192I; 2: LTR72/G192II; 3: LTR72/G192; 4: LTR72/G192 gene amplified by PCR; 5: double enzyme digestion identification of recombinant plasmid; 6: DL 2000.

Figure 3A shows LTK63/G192 induced expression. Among them, 1: after induction; 2: before induction; 3: protein marker.

Figure 3B shows LTK63/G192 protein purification. Among them, 1: protein marker; 2: purified LTK63/G192 protein; 3: miscellaneous protein.

Figure 3C shows the Western-Blot assay. Among them, 1: LTK63/G192; 2: LTR72/G192; 3: protein marker.

Fig. 4 shows the in vivo toxicity test results of Patent-mouse of LT and LT mutants.

The G/C value of PBS group was 0.071±0.0057; LT, 0.101±0.0035; LTK63, 0.073±0.0057; LTR72, 0.081±0.0084; LTG192, 0.091±0.0053, LTK63/R72, 0.075±0.0031 ; LTK63/G192, 0.080±0.0027; LTR72/G192, 0.081±0.0027 The error is ±s s, n=5.

Figure 5 shows the results of ELISA detection of antibody levels. Figure 5A shows the detection of serum Anti-NDV IgG; Figure 5B shows the detection of nasal wash Anti-NDV IgA; wherein,

a: NDV; b: NDV+LT; c: NDV+LTK63; d: NDV+LTR72; e: NDV+LTG192; f: NDV+LTK63/G192; g: NDV+LTR72/G192; R72.

第25天采样；

第35天采样。"□" Sampling on the 0th day; "■" Sampling on the 11th day;

Sampling on the 25th day;

Sampled on day 35.

Detailed ways

In the research, the present inventor unexpectedly found that the 63rd position of Escherichia coli heat-sensitive enterotoxin was mutated to K and the 192nd position was mutated to G (LTK63/G192), or the 72nd position of Escherichia coli heat-sensitive enterotoxin was mutated into R and the After the 192 position is mutated to G (LTR72/G192), the toxicity of the obtained Escherichia coli heat-sensitive enterotoxin mutant is significantly reduced, and it has excellent immune adjuvant activity. In addition, due to the double-site mutation of Escherichia coli heat-sensitive enterotoxin, the obtained mutant is less likely to undergo back mutation and has a more stable effect as an adjuvant.

The present inventors designed various single-site mutations or multi-site mutations for Escherichia coli heat-sensitive enterotoxin protein A subunit, and conducted in-depth research on these mutation methods. After toxicity analysis and enzyme activity test, the biological activity of each mutant was tested, and it was confirmed that the toxicity of the two mutants, LTK63/G192 and LTR72/G192, was significantly reduced, and the mucosal adjuvant activity was excellent. It was confirmed that serum antibodies and local secreted antibodies could be significantly increased by immunizing mice through the same route, so the mutants LTK63/G192 and LTR72/G192 can be developed as mucosal adjuvants for animal vaccines.

In the present invention, unless otherwise stated, the terms "Escherichia coli heat-sensitive enterotoxin mutant protein", "Escherichia coli heat-sensitive enterotoxin mutant polypeptide", "Escherichia coli heat-sensitive enterotoxin mutant", "Escherichia coli heat-labile enterotoxin "Double mutant" or "LT mutant" can be used interchangeably, both refer to the amino acid sequence corresponding to the heat-sensitive enterotoxin of wild-type Escherichia coli, the 63rd position is mutated to K, and the 192nd position is mutated to G; or For the amino acid sequence corresponding to wild-type Escherichia coli heat-sensitive enterotoxin, the 72nd position is mutated to R, and the 192nd position is mutated to G; or for the amino acid sequence corresponding to wild-type Escherichia coli heat-sensitive enterotoxin, A sequence in which the 63rd position is mutated to K, the 72nd position is mutated to R and the 192nd position is mutated to G. Of course, amino acid equivalent substitutions can also occur at some inactive related sites, for example, certain amino acids are replaced by amino acids with similar or similar properties.

As used herein, "isolated" means that the material is separated from its original environment (if the material is native, the original environment is the natural environment). For example, polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances that exist together in the natural state .

As used herein, "Escherichia coli heat-sensitive enterotoxin mutant protein or polypeptide" means that the E. coli heat-sensitive enterotoxin mutant protein does not substantially contain other proteins, lipids, carbohydrates or other substances naturally associated therewith. Those skilled in the art can purify the protein using standard protein purification techniques. Proteins of the invention can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast) using recombinant techniques.

As a preferred mode of the present invention, the Escherichia coli heat-sensitive enterotoxin mutant protein includes A subunit, and the amino acid sequence of this subunit is relative to the sequence shown in SEQ ID NO: 2 (its coding sequence is as shown in SEQ ID NO: 1 ), which is K at position 63 and G at position 192; or R at position 72 and G at position 192.

As a more preferred mode, the Escherichia coli heat-sensitive enterotoxin mutant protein also includes a B subunit (SEQ ID NO: 4, its coding sequence is as SEQ ID NO: 3). More preferably, the protein is basically composed of Escherichia coli heat-sensitive enterotoxin A subunit and B subunit. Optimally, the protein is basically composed of one A subunit and five B subunits, and the protein with this structure has a more ideal immune adjuvant effect.

Methods for introducing mutations into wild-type E. coli heat-sensitive enterotoxin protein sequences are known to those skilled in the art. For example, by designing primers containing mutation sites to PCR amplify the coding sequence of the wild-type Escherichia coli heat-sensitive enterotoxin protein, screen the gene product introduced into the mutation from the amplified product, and use the gene product to generate a mutant protein . In addition, proteins containing mutation sites in the sequence can also be synthesized artificially.

The present invention also includes polynucleotides encoding said proteins. A polynucleotide of the invention may be in the form of DNA or RNA. DNA can be single-stranded or double-stranded. The term "polynucleotide encoding a protein" may include a polynucleotide that encodes the polypeptide, or a polynucleotide that also includes additional coding and/or non-coding sequences.

The polypeptides and polynucleotides of the invention are preferably provided in isolated form, more preferably purified to homogeneity.

The nucleotide full-length sequence or its fragments of the Escherichia coli heat-sensitive enterotoxin mutant protein of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed according to the polynucleotide sequence encoding the protein, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art can be used. As a template, related sequences are amplified. When the sequence is long, it is often necessary to carry out two or more PCR amplifications, and then splice together the amplified fragments in the correct order.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.

In addition, related sequences can also be synthesized by artificial synthesis, especially when the fragment length is relatively short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them.

At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art. The method of amplifying DNA/RNA using PCR technique is preferably used to obtain the polynucleotide of the present invention.

The present invention also relates to a vector containing the polynucleotide of the present invention, a host cell produced by genetic engineering using the vector of the present invention or the coding sequence of the protein of the present invention, and a method for producing the polypeptide of the present invention through recombinant technology.

By conventional recombinant DNA technology (Science, 1984; 224:1431), the polynucleotide sequence of the present invention can be used to express or produce recombinant Escherichia coli heat-sensitive enterotoxin mutant protein. Generally speaking, there are the following steps:

(1). Use the polynucleotide (or variant) encoding Escherichia coli heat-sensitive enterotoxin mutant protein of the present invention, or transform or transduce a suitable host cell with a recombinant expression vector containing the polynucleotide;

(2). Host cells cultured in a suitable medium;

(3). Isolate and purify protein from culture medium or cells.

Preferably, the Escherichia coli heat-sensitive enterotoxin mutant protein is expressed in Escherichia coli, and since the Escherichia coli heat-sensitive enterotoxin itself is derived from Escherichia coli, the protein obtained by expression in Escherichia coli has good activity.

In a preferred example of the present invention, the expression of the mutant protein of the present invention is improved, so as to obtain the LT mutant protein with substantially the same ratio and spatial structure of the A subunit and B subunit of the natural state. The inventors found that after the A subunit and the B subunit are expressed in bacteria, the A subunit basically forms an inclusion body, and because the B subunit is small, part of it forms an inclusion body, and the other part is expressed in a soluble form. In order to ensure that the A and B subunits are not separated or the ratio is out of balance, the bacterial pellet obtained by centrifugation is directly resuspended with a denaturant (preferably NTA0), and the supernatant is collected by centrifugation after thorough stirring. In this way, the A subunit and the B subunit are still together, and the ratio between the A subunit and the B subunit is not disturbed; after renaturation, the E. coli heat-labile enterotoxin structure is not destroyed. However, according to the existing routine operation, after the cell pellet is obtained by centrifugation, the bacteria-breaking treatment is performed first, and then the inclusion body is treated with a denaturant, which will remove most of the B subunits, so that the protein configuration obtained subsequently is consistent with the natural one. The status is different.

In the present invention, the polynucleotide sequence can be inserted into an expression vector. The term "expression vector" refers to bacterial plasmids, yeast plasmids or other vectors well known in the art. Any plasmid and vector can be used as long as it can be replicated and stabilized in the host. An important feature of expression vectors is that they usually contain an origin of replication, a promoter, marker genes, and translational control elements.

Methods well known to those skilled in the art can be used to construct recombinant expression vectors containing the polynucleotide sequences of the present invention and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology and the like. Said DNA sequence can be operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis.

In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as tetracycline or ampicillin resistance for E. coli.

Vectors containing the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences can be used to transform appropriate host cells so that they can express proteins. The host cell can be a prokaryotic cell, such as a bacterial cell.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the _CaCl2 method using procedures well known in the art. Another method is to use _MgCl2 . Transformation can also be performed by electroporation, if desired.

The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. The medium used in the culture can be selected from various conventional media according to the host cells used. The culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time.

Recombinant proteins can be isolated and purified by various isolation methods, which are well known to those skilled in the art, by taking advantage of their physical, chemical and other properties. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, supertreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

In a preferred example of the present invention, the inventors used overlap extension PCR amplification technology to obtain the entire gene fragments of Escherichia coli heat-labile enterotoxin double mutants LTK63/G192 and LTR72/G192 in vitro, and the results of enzyme digestion and sequencing showed that the constructed The reading frames of the expression vectors pET30a-LTK63/G192 and pET30a-LTR72/G192 were correct, and the corresponding amino acids were substituted. The induced expression products were detected by SDS-PAGE, and each mutant expressed two protein bands of about 33.0Ku and 13.0Ku, which were consistent with the molecular weights of LTA and LTB subunits; His antibody produces a specific immune reaction. ADP-ribosyltransferase activity test analysis showed that the protease activity of the two mutants was lower than that of the wild-type LT protein, but some enzyme activities were still retained. According to the Patent-mouse toxicity test, the toxicity of LTK63/G192 and LTR72/G192 mutant proteins is very low. The two proteins were used as adjuvants for NDV to immunize mice by nasal drop, and the results showed that both LTK63/G192 and LTR72/G192 had higher adjuvant activity.

The present invention also provides the use of the Escherichia coli heat-sensitive enterotoxin mutant protein for preparing an adjuvant for mucosal immunity, which has the characteristics of stable performance, high antibody level induced by the body, and low toxicity.

In the Escherichia coli heat-sensitive enterotoxin mutant, the B subunit has immune adjuvant activity because it can regulate the combination of LT and GM1 on the cell membrane. However, because the B subunit itself has a small molecular weight, it is used alone as an adjuvant , after entering the body, it is easy to cause the reduction or disappearance of immunogenicity, and the effect is not ideal, while the combination of B subunit and A subunit with reduced toxicity has good activity as an immune adjuvant and has high immunogenicity.

Therefore, the present invention also provides a composition for mucosal immunity, which contains a safe and effective amount of the Escherichia coli heat-sensitive enterotoxin mutant protein of the present invention, and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. The compositions of the invention may be formulated in any form suitable for mucosal administration. The content of Escherichia coli heat-sensitive enterotoxin mutant protein in the composition may be, for example, 0.0001-20wt%; more preferably 0.001-10wt%.

When using the composition, a safe and effective amount of E. coli heat-sensitive enterotoxin mutant protein is administered to the mammal, wherein the safe and effective amount is usually at least about 1 microgram/kg body weight, and in most cases no more than about 8 mg /kg body weight, preferably the dose is about 1 microgram/kg body weight to about 1 mg/kg body weight. Of course, the specific dose should also consider factors such as the way of administration, the physical condition of the subject, and these are within the skill of skilled physicians.

In addition, the composition also contains: an effective amount of vaccine, the content of which in the composition is, for example, 0.0001-20wt%; more preferably 0.001-10wt%. Therefore, with the assistance of the Escherichia coli heat-sensitive enterotoxin mutant protein of the present invention, a good vaccine immune effect can be produced.

The main advantages of the present invention are:

(1) It was confirmed that the two mutants, LTK63/G192 and LTR72/G192, had significantly reduced toxicity and excellent mucosal adjuvant activity, which was significantly better than LTK63/G72.

(2) The expression process of the LT mutant is optimized, so that the obtained protein is closer to its natural existence mode and has good activity.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods not indicating specific conditions in the following examples are usually according to conventional conditions such as Sambrook et al., molecular cloning: the conditions described in the laboratory guide (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's suggested conditions. Percentages and parts are by weight unless otherwise indicated.

Materials and Methods

1. Strains and plasmids

Strains and plasmids: Using P1/P2 as primers and Escherichia coli producing Escherichia coli heat-sensitive enterotoxin as a template, the wild LT gene was amplified by PCR, digested with NcoI and SalI, and then ligated into pET30a (Novagen) that had undergone the same digestion , the plasmid pET30a-LT was obtained.

As the bacterial strain, conventional Escherichia coli DH5α, BL21(DE3) was used.

2. Experimental animals

Kunming female mice aged 6 to 8 weeks were provided by the Experimental Animal Department of Fudan University School of Medicine, and Newcastle disease low-virulence live vaccine (Lasota) was purchased from Shanghai Songjiang Biopharmaceutical Co., Ltd.

3. Mutant expression vector construction

(1) Primer design

Refer to the registered LT whole gene sequence, design primer pair P1/P2 for amplifying the LT whole gene, and add NcoI and SalI restriction enzyme sites (indicated by the framed part) and protective bases at both ends of the primers; The primer P3/P4 was designed with the 63rd amino acid site as the center, so that the 63rd amino acid codon was converted from the Ser codon TCT to the Lys codon AAA (underlined); the nucleotide centered on the 72nd amino acid of LT The sequence is based on the design of the mutation primer pair P5/P6, and the 72-position GCA is replaced by CGT (underlined); the P7/P8 mutation primer pair is designed based on the nucleotide sequence centered on the 192nd amino acid of LT, and the 192-position AGA was replaced by GGA (underlined), and the primers were synthesized by Shanghai Sangong.

P3: 5'-CGGATATGTTCCACT AAA CTTAGTTTGAGAAGTGCTC-3' (SEQ ID NO: 7);

P4: 5'-GAGCACTTCTCAAACTAAG TTT AGTGGAAACATATCCG-3' (SEQ ID NO: 8);

P5: 5'-GAGAAGTGCTCACTTA CGT GGACAGTCTATATTATTATCAGG-3' (SEQ ID NO: 9);

P6: 5'-CCTGATAATATAGACTGTCC ACG TAAGTGAGCACTTCTC-3' (SEQ ID NO: 10);

P7: 5'- GGTTGTGGAAATTCATCAGGAACAATCACAGGTGATACTTG -3' (SEQ ID NO: 11);

P8: 5'- CAAGTATCACCTGTGATTGTTCCTGATGAATTTCCACAACC -3 (SEQ ID NO: 12).

(2) Construction of LTK63 mutant gene expression vector

a. Preparation of mutant gene fragments I and II

Add dNTPs, Ex Taq, 10× buffer, corresponding primer pair, sterile water, and template pET30a-LT to the 100 μl reaction system. The primer pair used for the amplification of the LTK63 gene fragment I is P1/P4, and the primer pair used for the amplification of the fragment II is P2/P3. After the reaction conditions are completed, the PCR product is recovered.

b. Preparation of mutant LTK63 gene

Mix the PCR products in a., add dNTPs, Ex Taq, 10× buffer, and sterile water for 8 PCR cycles, then add dNTPs, Ex Taq, 10× buffer, sterile water and primer pair P1/P2. A new PCR cycle, the reaction is completed, and the PCR product is recovered.

c. Construction of expression vector and identification of recombinant plasmid

The above PCR product and pET30a were digested with NcoI and SalI respectively, recovered from the rubber, and joined with DNA Ligation Kit at an appropriate molar ratio. The ligation product was transformed into DH5α. The clones were picked to extract the plasmids and identified by PCR and enzyme digestion. Sequence the positive recombinant plasmid to verify whether the reading frame and mutation site are correct.

(3) LTR72 mutant gene expression vector construction

The primer pair used for fragment I amplification was P1/P5, the primer pair used for fragment II amplification was P2/P6, and the rest of the operations were constructed as LTK63 mutant gene expression vector.

(4) Construction of LTK63/G192 mutant gene expression vector

a. Preparation of mutant gene fragments I and II

Add dNTPs, Ex Taq, 10× buffer, corresponding primer pair, sterile water, and template pET30a-LTK63 into the 100 μl reaction system. The primer pair used for the amplification of the LTK63/G192 gene fragment I is P1/P8, and the primer pair used for the amplification of the fragment II is P2/P7. After the reaction is complete, the PCR product is recovered.

b. Preparation of the 63rd and 192 mutant genes (LTK63/G192)

Mix the PCR products obtained above, add dNTPs, Ex Taq, 10× buffer, and sterile water for 8 PCR cycles, then add dNTPs, Ex Taq, 10× buffer, sterile water and primer pair P1/P2 A new PCR cycle, the reaction is completed, and the PCR product is recovered.

c. Construction of expression vector and identification of recombinant plasmid

The above PCR product and pET30a were digested with NcoI and SalI respectively, recovered by tapping the gel, and joined with a conventional DNA ligation kit for ligation. The ligation product was transformed into Escherichia coli DH5α. Single clones were picked and plasmids were extracted, and identified by PCR and enzyme digestion by conventional methods. The positive recombinant plasmid was sequenced to verify the reading frame and mutation site, so as to obtain the recombinant expression vector pET30a-LTK63/G192 with the correct mutation site.

(5) Construction of LTR72/G192 mutant gene expression vector

Except for corresponding changes in the template and primers, the rest of the operations were constructed as LTK63/G192 mutant gene expression vectors.

4. Expression, purification and Western Blot detection of recombinant protein

Express

The recombinant plasmid was transformed into BL21(DE3). After PCR identification, the protein was induced to express with IPTG, the culture supernatant was removed by centrifugation of the bacterial solution, and the bacteria were resuspended using NTA0 (20mmol/L Tris-HCl, 0.5mol/L NaCl, 10% glycerol) Precipitate, sonicate the cells, stir overnight with a magnetic stirrer at 4°C, and collect the supernatant by centrifugation.

The characteristic of this experiment is that: LT is composed of A subunit and B subunit, and A and B subunits are expressed to form a complete LT protein. Therefore, the bacterial pellet obtained by centrifugation in this experiment was directly resuspended in Urea-NTAO, stirred overnight with a magnetic stirrer at 4°C, and the supernatant was collected by centrifugation. In this way, the A subunit and the B subunit are still together, and the ratio between the A subunit and the B subunit is not disturbed, so that after renaturation, the E. coli heat-labile enterotoxin structure is not destroyed.

Refolding adopts conventional methods, and dialyzes in NTA0 containing 2M urea.

purification

Pack the column with Ni-NTA Resin and purify it with FPLC system. The purified protein was refolded by dialysis at 4°C for 24 hours, then dialyzed in PBS, and quantified by BCA method.

Western blot detection

The purified protein was subjected to conventional SDS-PAGE electrophoresis, and transferred to a nitrocellulose membrane, and anti-CT antibody and His-tag antibody (both purchased from NEWLANBORS Company) were used as primary antibodies to detect the protein of the sample.

5. Detection of ADP-ribosyltransferase activity of recombinant protein

ADP-ribosyltransferase activity detection is based on literature Soman G et al., Use of Substituted (Benzylidineamino)guanidines in the Study of Guanidino Group Specific ADP-ribosyltransferase[J]; Biochemistry, 1986, 25: 4113-4119 and De Haan et al., Mucosal immunogenicity and Adjuvant activity of the recombinant Asubunit of the Escherichia coli heat-labile enterotoxin[J]. Immunology, 1999, 97(4): 706-713 method.

Use aminoguanidine bicarbonate and p-diethylaminobenzaldehyde to react to generate DEA-BAG (p-diethylamino-(benzylidineamino)guanidine), and use 8 known DEA-BAG concentrations to detect the light absorption at 355nm , draw the standard concentration-light absorption value curve. 750 ng of each protein was treated with trypsin and placed in a water bath at 37°C for 1 hour. The reaction was inhibited with soybean trypsin inhibitor. Add an equal amount of DEA-BAG solution, start the reaction with NAD, and bathe in water at 30°C for 2 hours. Use an equal amount of DOWEX-50W resin to adsorb unreacted DEA-BAG. After filtration, the light absorption of each reaction filtrate was detected at 355 nm, and each protein was replicated 3 times. Calculate the concentration of DEA-BAG in the reaction solution according to the standard curve, and calculate the amount of DEA-BAG consumed. Taking the amount of DEA-BAG consumed by LT as 100% enzyme activity, the relative enzyme activity of each protein was calculated.

6. Toxicity detection of recombinant protein

The Patent-mouse toxicity detection test was carried out according to the method of Feng Qiang et al., Expression, Purification and Preservation Strategy of Escherichia coli Heat-labile Enterotoxin [J]; Acta Bioengineering Sinica, 2003, 19(5): 532-537. The obtained data were tested for significant difference by t-test, P>0.05 was not significant, P≤0.01 was extremely significant, and 0.01<P≤0.05 was significant.

7. Detection of protein adjuvant activity

a. Mouse immunization experiment

Female mice aged 5-6 weeks were randomly divided into 8 groups, 5 mice/group. A group of Newcastle disease vaccine (dissolved in 0.1M PBS) was immunized with only 5 feathers (refer to the vaccine instruction manual), and the other groups were immunized with 5 feathers of Newcastle disease vaccine and 4 μg of various toxin proteins (dissolved in 0.1M PBS). 0.1M in PBS) immunization. The first, second and third immunizations were carried out on days 1, 12 and 26 respectively. On days 0, 11, 25, and 35, blood was collected by tail docking, and the nose was washed with 500 μl of PBS (0.1% BSA). Blood samples were left overnight at 4°C, centrifuged at 5000rpm/min for 5min to collect serum for detection. Store at -20°C.

b. Indirect ELISA detection of antibody levels in different treatment groups

The Newcastle disease LaSota strain virus was used to coat the ELISA reaction plate (100 μl/well), and the serum IgG and IhA antibody levels in the nasal wash were detected by conventional methods.

Example

Example 1 PCR amplification and restriction analysis of expression vector construction

Using the previously constructed mutant expression vectors pET30a-LTK63 and pET30a-LTR72 as templates and P1/P4 as primers to amplify the LTK63/G192I and LTR72/G192I genes with a size of about 600 bp; use P2/P3 as primers to amplify The LTK63/G192II and LTR72/G192II genes with a size of about 530bp were obtained. Using each pair of fragments I and II as templates and using P1/P2 as primers, LTK63/G192 and LTR72/G192 of about 1100 bp were amplified by overlapping region amplification gene splicing method, and the sizes were similar to those expected.

The sequencing results showed that the 63rd amino acid of the LT gene fragment in pET30a-LTK63/G192 was mutated from Ser to Lys, and the 192nd amino acid was mutated from Arg to Gly; the 72nd amino acid codon of the LT gene fragment in pET30a-LTR72/G192 Ala was mutated to Arg, and the 192nd amino acid codon was mutated from Arg to Gly.

The agarose gel electrophoresis analysis of the expression vectors pET30a-LTK63/G192 and pET30a-LTR72/G192 is shown in Figure 1 and Figure 2 .

Induced expression, purification and Western-Blot detection of embodiment 2 recombinant bacteria

The mutant strains with the correct reading frame were inoculated into liquid medium respectively, and their expression was induced with IPTG at a final concentration of 1 mmol/L. Both mutants expressed two protein bands at about 33.0Ku and 13.0Ku; recombinant The expressed protein was purified with conventional Ni-NTA resin, eluted with imidazole, and two bands at about 33.0Ku and 13.0Ku were detected by electrophoresis, and the anti-His-tag antibody was used as the primary antibody to detect the recombinant protein. The expression of LTK63/G192 protein is shown in Figure 3A, the SDS-PAGE of LTK63/G192 protein purification is shown in Figure 3B, and the Western-Blot detection of LTK63/G192 protein and LTR72/G192 protein is shown in Figure 3C.

Example 3 Detection of ADP-ribosyltransferase activity

Use DEA-BAG as the substrate, start the reaction with NAD, measure the light absorption value at 355nm after the reaction is terminated, calculate the substrate consumption according to the standard formula, take the substrate consumed by LT as the enzyme activity 100%, and calculate For the relative enzyme activities of other proteins, an equal volume of protein-free PBS was used as a control. The results are shown in Table 1.

Table 1 ADP-ribosyltransferase activity detection results

toxin protein ADP-ribosyltransferase (%) Control (PBS) 6.54 LT 100 LTK63 11.71 LTR72 12.06 LTG192 8.71 LTK63/G192 8.76 LTR72/G192 8.78

It can be seen from the above table that the ADP-ribosyltransferase activity of LTK63/G192 and LTR72/G192 is lower than that of LTK63 and LTR72.

The toxicity detection of embodiment 4 LT mutant

Feed the mice (Patent-mouse) with PBS, wild-type LT protein and each mutant protein respectively, take the ratio of the intestinal weight to the body weight, and calculate the mean and standard deviation of each group. The wild-type LT was tested by t-test, and the results are shown in Table 2 and Figure 4.

Table 2 Patent-mouse in vivo toxicity detection t-test results

The results showed that the toxicity of each mutant was significantly reduced compared with wild-type LT.

Example 5 Adjuvant Activity Research

Mice in each group were immunized with Newcastle disease vaccine (NDV), or Newcastle disease vaccine and each toxin protein on day 1, 12 and 26 respectively for the first, second and third times. On days 0, 11, 25, and 35, blood was collected by docking the tail to collect serum, and at the same time, 500 μl of PBS (0.1% BSA) was used to wash the nose. The IgG of each serum sample and the IgA of nasal wash samples were detected by ELISA, and the OD value was read under 450nm light wave. After the third immunization, the readings of the toxin-added protein in each group and the readings of the NDV-immunized group were used for t-test. P>0.05 the difference is not significant, 0.01<P<0.05 the difference is significant, P<0.01 the difference is extremely significant. The results are shown in Figure 5 and Table 3-Table 4.

Table 3: t-value test of IgG levels between the toxin protein+NDV group and the NDV group after three immunizations

Table 4: t-value test of IgA levels of each toxin-added protein+NDV group and NDV group after three immunizations

The results showed that compared with NDV alone, simultaneous use of LTK63/G192 or LTR72/G192 can significantly increase the IgG and IgA levels in animals, that is, they have good immune adjuvant activity; while LTK63/R72 is not significant.

Example 6 Composition

A composition for mucosal immunization, which contains: 4 μg of the aforementioned purified LT mutant protein LTR72/G192 as an adjuvant for mucosal immunization; 1-2 portions of Newcastle disease vaccine (dissolved in PBS medium, 0.5ml); and 0.5mL of 0.1M PBS.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

sequence listing

Claims (10)

1. an isolated Escherichia coli heat-sensitive enterotoxin mutant protein, characterized in that, the protein comprises A subunit and B subunit of the Escherichia coli heat-sensitive enterotoxin protein, and the amino acid sequence of the A subunit is relative to the wild The amino acid sequence of the A subunit of Escherichia coli heat-sensitive enterotoxin, its 63rd position is K and its 192nd position is G; or Its 72nd position is R, and its 192nd position is G. 2. The protein according to claim 1, characterized in that, the protein contains one A subunit of Escherichia coli heat-sensitive enterotoxin protein and five B subunits of Escherichia coli heat-sensitive enterotoxin protein. 3. An isolated nucleic acid, characterized in that said nucleic acid encodes the protein according to claim 1 or 2. 4. A carrier, characterized in that the carrier contains the nucleic acid according to claim 3. 5. A cell, characterized in that the cell contains the vector according to claim 4, or the nucleic acid according to claim 3 is integrated in its genome. 6. A method for producing the protein according to claim 1 or 2, characterized in that it comprises: cultivating the cell according to claim 5 under conditions suitable for expression, thereby expressing the protein according to claim 1 or 2 protein. 7. The method of claim 6, comprising: (1) expressing the A subunit and the B subunit of the heat-sensitive enterotoxin mutant protein of Escherichia coli in the cell; (2) Collect the cells of (1), resuspend the cells with a denaturant, and break the cells; (3) Mix the cell disruption product of (2), and collect the supernatant by centrifugation; (4) Separate the protein containing A subunit and B subunit from the supernatant obtained in (3), and refold to obtain the desired protein. 8. The use of the protein according to claim 1 or 2, characterized in that it is used to prepare an adjuvant for mucosal immunization. 9. A composition for mucosal immunization, characterized in that the composition contains: an effective amount of the protein according to claim 1 or 2 as an adjuvant for mucosal immunization. 10. The composition according to claim 9, characterized in that, the composition further comprises: an effective amount of vaccine for mucosal immunity.

Images