CN113265365B

CN113265365B - Method for efficiently expressing exogenous protein in escherichia coli expression system and application thereof

Info

Publication number: CN113265365B
Application number: CN202010096475.0A
Authority: CN
Inventors: 田克恭; 李鹏昊; 张许科
Original assignee: Pulaike Biological Engineering Co Ltd
Current assignee: Pulaike Biological Engineering Co Ltd
Priority date: 2020-02-17
Filing date: 2020-02-17
Publication date: 2023-10-17
Anticipated expiration: 2040-02-17
Also published as: CN113265365A

Abstract

The invention relates to an escherichia coli construction method for efficiently expressing exogenous proteins and application thereof, wherein the method comprises the following steps: step (1) BL21 (DE 3) lpxM gene knockout; step (2) inserting tig genes into the BL21 (DE 3) lpxM gene deletion strain obtained in the step (1); (3) constructing a recombinant vector pET28a-Rcodon; (4) Cloning the exogenous protein gene to the recombinant vector pET28a-Rcodon obtained in the step (3); (5) Transforming the recombinant vector obtained in the step (4) into the strain obtained in the step (2); and (6) harvesting the exogenous protein. The method of the invention can make the foreign protein efficiently expressed, can obviously improve the expression quantity of the foreign protein by the conventional expression of the escherichia coli, greatly reduce the endotoxin content, can be applied to the efficient expression of various foreign proteins, can be widely applied to various veterinary vaccine proteins including eukaryotic proteins, and has wide application range.

Description

Method for efficiently expressing exogenous protein in escherichia coli expression system and application thereof

Technical Field

The invention relates to a method for efficiently expressing exogenous proteins in an escherichia coli expression system and application thereof. The invention belongs to the field of biological pharmacy, and relates to genetic engineering modification and application of escherichia coli.

Background

Coli (e.coli) prokaryotic expression system is widely used for the expression of heterologous proteins and the research of related subunit vaccines due to the advantages of convenient operation, high expression efficiency, low cost and the like, however, the e.coli prokaryotic expression system has some defects, which severely restrict the efficacy and further application of the expression system. E.coli is a gram-negative bacterium, and its Lipopolysaccharide (LPS) structure can cause a relatively serious endotoxin reaction clinically, and proteins expressed by using e.coli as a host generally face the difficulty of endotoxin removal, which increases the difficulty and cost of downstream protein purification processes to some extent. E.coli lacks enzymes required for efficient folding of the protein itself and the oxidation state intracellular environment required for disulfide bond formation, and the protein is prone to inclusion body formation or degradation after misfolding. In addition, E.coli, which is a prokaryotic expression host strain, cannot synthesize a part of transfer RNA corresponding to rare codons, and therefore, when a heterologous protein, particularly a eukaryotic-derived protein, is expressed, there are phenomena of low transcription flux and weak protein expression.

At present, no high-efficiency E.coli host strain with low toxicity and high solubility is available for expressing the foreign proteins, and a high-efficiency expression method which is applicable to different foreign proteins and is easy to operate is developed, so that the biosafety of the escherichia coli subunit vaccine is improved, and the method has wide market application prospect.

Disclosure of Invention

In order to solve the problems, the invention provides an escherichia coli construction method for efficiently expressing exogenous proteins, wherein the method comprises the following steps: step (1) BL21 (DE 3) lpxM gene knockout; step (2) inserting tig genes into the BL21 (DE 3) lpxM gene deletion strain obtained in the step (1); (3) constructing a recombinant vector pET28a-Rcodon; (4) Cloning the exogenous protein gene to the recombinant vector pET28a-Rcodon obtained in the step (3); (5) Transforming the recombinant vector obtained in the step (4) into the strain obtained in the step (2); and (6) harvesting the exogenous protein.

In the step (1), the left and right arms of the lpxM gene are amplified by using genomic DNA of BL21 (DE 3) strain as a template, and the left and right arms are overlapped by overlap PCR; constructing pTargetF-sgRNA1 and pTargetF-sgRNA2 vectors, and electrically transforming BL21 (DE 3)/pCas competent cells together with the left and right arms; extracting genome DNA of the transformant as a template for PCR amplification; the transcription level of the lpxM gene was semi-quantitatively analyzed by RT-PCR to verify that the lpxM gene deletion was successful.

In step (2), synthesizing a tig gene into a pET28a vector to provide a tig gene expression cassette; amplifying a tig-cassette expression cassette containing a T7 promoter and a terminator by taking a pET28a-tig vector as a template; amplifying left and right arms of the lpxM gene using genomic DNA extracted from BL21 (DE 3) ΔlpxM mutant strain (i.e., BL21 (DE 3) lpxM gene deleted strain) as a template, and connecting the left and right arms of lpxM to both ends of the tig-cassette fragment by overlap PCR; the recombined fragment and pTargetF-sgRNA1 and pTargetF-sgRNA2 vectors are used for electrotransformation of BL21 (DE 3) DeltalpxM/pCas competent cells; extracting genome DNA of the transformant as a template for PCR amplification, and taking the genome DNA of BL21 (DE 3) and BL21 (DE 3) DeltalpxM as a control; semi-quantitative analysis of the transcript level of the tig gene was performed by RT-PCR in order to verify the success of the tig gene integration expression.

In step (3), synthesizing a gene sequence comprising tRNA elements into a pUC57 vector to construct a pUC57-Rcodon vector; amplifying skeleton parts except for ori and Lac I element intervals by taking pET28a as a template, wherein the size of the skeleton parts is about 4300 bp; amplifying a sequence fragment containing seven tRNA coding genes by using pUC57-Rcodon as a template, wherein the size of the fragment is about 2800bp, and two ends of the fragment respectively have 20bp motifs homologous with pET28a skeletons; recombining the skeleton fragment of pET28a and the fragment containing tRNA coding gene, and screening transformant after transforming DH5 alpha competent cells by the recombination system; the transformant plasmid was extracted and sequenced to verify that the recombinant vector (pET 28 a-Rcodon) was correct.

In the step (4), the exogenous protein sequence and the pET28a-Rcodon recombinant vector are subjected to double digestion, and DH5 alpha competent cells are transformed after the digestion products are connected; after extracting the transformant plasmid, double enzyme digestion is carried out to obtain a target fragment so as to verify that the recombinant vector pET28 a-Rcodon-foreign protein is successfully constructed.

In the step (5), the recombinant vector pET28 a-Rcodon-exogenous protein is converted into BL21 (DE 3) delta lpxM tig obtained in the step (2) ⁺ Strains.

The method of the invention can make the foreign protein efficiently expressed, can obviously improve the expression quantity of the foreign protein by the conventional expression of the escherichia coli, greatly reduce the endotoxin content, can be applied to the efficient expression of various foreign proteins, can be widely applied to various veterinary vaccine proteins including eukaryotic proteins, and has wide application range.

In the method for constructing escherichia coli with high-efficiency expression of exogenous proteins according to the present invention, the exogenous proteins in the step (4) include african swine fever virus CD2v protein, avian adenovirus Penton protein, avian adenovirus Fiber-2 protein, avian egg drop syndrome virus Penton protein, avian egg drop syndrome virus tFiber protein, chicken infectious bursal disease virus VP2 protein, porcine circovirus 3 Cap protein, porcine circovirus 2 Cap protein, porcine pseudorabies virus gB protein, porcine parvovirus VP2 protein, swine fever virus E2 protein, bovine infectious rhinotracheitis virus gB protein, bovine infectious rhinotracheitis virus gD protein, foot-and-mouth disease virus VP0 protein, foot-and-mouth disease virus VP3 protein, foot-and-mouth disease virus VP1 protein, rabbit pestivirus VP60 protein, schistosome GALE protein, and japanese schistosome Wnt5 protein.

The invention also relates to the escherichia coli which is prepared by the method and is used for efficiently expressing the exogenous protein.

The invention also relates to a method for efficiently expressing the exogenous protein, wherein the method uses the escherichia coli to express the exogenous protein.

The invention also relates to the application of the method in preparing exogenous proteins.

The invention also relates to a vaccine composition prepared from the prepared exogenous protein, and the exogenous protein prepared by the method is added with a pharmaceutically acceptable carrier to prepare a subunit vaccine composition.

The exogenous protein prepared by the preparation method has no adverse reaction on the growth and development of animals in terms of biological safety, immunogenicity and immune efficacy, and can be used for preparing subunit vaccines.

As an embodiment of the present invention, in the method for preparing a vaccine according to the present invention, the pharmaceutically acceptable carrier includes an adjuvant, and the adjuvant includes: (1) Mineral oil, aluminium gel adjuvant, saponin, alfulidine, DDA; (2) A water-in-oil emulsion, an oil-in-water emulsion, and a water-in-oil-in-water emulsion; or (3) a polymer of acrylic acid or methacrylic acid, a copolymer of maleic anhydride and an alkenyl derivative; and one or more of RIBI adjuvant system, block co-polymer, SAF-M, monophosphoryl lipid A, avridine lipid-amine adjuvant, escherichia coli heat labile enterotoxin, cholera toxin, IMS 1314, muramyl dipeptide, montanide ISA 206, gel adjuvant; preferably, the saponin is Quil A, QS-21, GPI-0100;

the adjuvant content is 5% -70% V/V.

The adjuvant may be present in any amount selected from 5% V/V, 6% V/V, 7% V/V, 8%V/V, 9%V/V, 10% V/V, 15% V/V, 20% V/V, 25% V/V, 30% V/V, 35% V/V, 40% V/V, 45% V/V, 50% V/V, 55% V/V, 60% V/V, 65% V/V, 66% V/V, 67% V/V, 70% V/V.

The vaccine compositions of the present invention may be formulated using available techniques, preferably together with a pharmaceutically acceptable carrier. For example, the oil may help stabilize the formulation and additionally act as a vaccine adjuvant. The oil adjuvant can be of natural origin or obtained by artificial synthesis. The term "adjuvant" refers to a substance added to the compositions of the present invention to increase the immunogenicity of the composition. Known adjuvants include, but are not limited to: (1) aluminum hydroxide, saponins (Saponine) (e.g., quilA), alfutidine, DDA, (2) polymers of acrylic acid or methacrylic acid, polymers of maleic anhydride and alkenyl derivatives, (3) vaccines may be formulated as oil-in-water, water-in-oil, or water-in-oil-in-water emulsions, or (4) Montanide ^TM Gel。

In particular, the emulsion may be based on light liquid paraffin oils, isoprenoid oils, such as squalane or squalene; the oligomerization of olefins, particularly isobutylene or decene produced oils, esters of acids or alcohols with linear alkyl groups, more particularly vegetable oils, ethyl oleate, propylene glycol di (caprylate/caprate), glycerol tri (caprylate/caprate), propylene glycol dioleate; branched fatty acid esters or esters of alcohols, in particular isostearic acid esters. The oil is used with an emulsifier to form an emulsion. The emulsifier is preferably a nonionic surfactant, in particular a polyoxyethylenated fatty acid (e.g. oleic acid), sorbitan, mannitol (e.g. sorbitan oleate), glycerol, polyglycerol, esters of propylene glycol and optionally ethoxylated oleic acid, isostearic acid, ricinoleic acid, hydroxystearic acid, ethers of fatty alcohols and polyols (e.g. oleyl alcohol), polyoxypropylene-polyoxyethylene block copolymers, in particular PluronicR, in particular L121 (cf. Hunter et al, 1995, "The Theory and Practical Application ofAdjuvants" (Steward-Tull, D.E.S. Main) John Wiley andSons, NY,51-94; todd et al, vaccine,1997, 15, 564-570).

In particular, the acrylic or methacrylic polymer is crosslinked by polyalkenyl ethers of sugars or polyols. These compounds are known as carbomers.

The amount of adjuvant suitable for use in the compositions of the present invention is preferably an effective amount. By "effective amount" is meant that amount necessary or sufficient for the adjuvant to exert its immunological effect in the host when administered in combination with the antigen of the invention without causing undue side effects. The exact amount of adjuvant to be administered will vary depending on factors such as the ingredients used and the type of disease being treated, the type and age of the animal to be treated, the manner of administration, and other ingredients in the composition.

The vaccine composition of the invention, preferably a subunit vaccine composition, may further comprise one or more of the group consisting of: drugs, immunostimulants (e.g., interferon-alpha, interferon-beta, interferon-gamma, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), and interleukin 2 (IL 2)), antioxidants, surfactants, colorants, volatile oils, buffers, dispersants, propellants, and preservatives. To prepare such compositions, methods well known in the art may be used.

The vaccine compositions, preferably subunit vaccine compositions, according to the present invention may be prepared in oral or non-oral dosage forms.

Preferred are non-oral dosage forms that can be administered by intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal or epidural routes.

Detailed Description

The advantages and features of the present invention will become more apparent from the following description of the embodiments. These examples are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions fall within the scope of the present invention.

The chemical reagents used in the examples of the invention are all analytically pure and purchased from the national drug group.

In order that the invention may be more readily understood, the invention will be further described with reference to the following examples. The experimental methods provided by the invention are conventional methods unless specified; the biological material, unless otherwise specified, is commercially available.

EXAMPLE 1 construction method of E.coli highly expressing infectious bursal disease Virus VP2 protein

1. Knock-out of the E.coli BL21 (DE 3) lpxM Gene

1. Design of synthetic primers

The genome sequence of lpxM (Gene accession number: CP 001509.3) based on the E.coli BL21 (DE 3) genome sequence in GeneBank was self-designed using Primer5.0 and was completed by Suzhou Jin Weizhi Biotech Co.

sgRNA-1-F：

ATATATACTAGTGGGACGTTTTGCCGGACGACGTTTTAGAGCTAGAAATAGC

sgRNA-2-F：

ATATATACTAGTGATGGATGATCTGTTAGAGGGTTTTAGAGCTAGAAATAGC

sgRNA-R：ATATATACTAGTATTATACCTAGGACTGAGCTAG

lpxM-L-F：TGCACTATGAAGTATGGATA

lpxM-L-R：

CCGCGCAATCGTATGATCATAACGTCCCAGCCGTGCCAGA

lpxM-L-Rt：

ATTTCGCGGGATCGAGATCTAACGTCCCAGCCGTGCCAGA

lpxM-R-F：

TCTGGCACGGCTGGGACGTTATGATCATACGATTGCGCGG

lpxM-R-Ft：

CTGAAAGGAGGAACTATATCATGATCATACGATTGCGCGG

lpxM-R-R：TAATGACGGCGTAGCAGCTG

lpxM-RT-F：GGCAAGGGCTGGAGATCATC

lpxM-RT-R：GTACCGACTG GATGAATGGT

2. Construction of BL21 (DE 3) ΔlpxM mutant

Amplifying left and right arms of the lpxM gene by taking genomic DNA of BL21 (DE 3) strain as a template, and overlapping the left and right arms by overlapping PCR; constructing pTargetF-sgRNA1 and pTargetF-sgRNA2 vectors, and electrically transforming BL21 (DE 3)/pCas competent cells together with the left and right arms; the genomic DNA of the transformant was extracted as a template for PCR amplification. The size of the fragment amplified by taking the genomic DNA extracted from the positive transformant as a template is about 950bp, which is about 640bp smaller than the control band. Semi-quantitative analysis of the transcription level of the lpxM gene was performed by RT-PCR, and as a result, the lpxM deletion mutant (BL 21 (DE 3) Δlpxm mutant) did not amplify any band, confirming that the lpxM gene deletion was successful.

2. Integrated expression of tig Gene on BL21 (DE 3) DeltalpxM genome

1. Design of synthetic primers

The tig gene (Genebank SEQ ID NO: ADY 57932.1) was synthesized into the pET28a vector by Suzhou Jin Weizhi Biotechnology Co., ltd, to provide a tig gene expression cassette.

tig-cassette-F：

TCTGGCACGGCTGGGACGTTAGATCTCGATCCCGCGAAAT

tig-cassette-R：

CCGCGCAATCGTATGATCATGATATAGTTCCTCCTTTCAG

tig-RT-F：GAAGCAGAAGACCGCGTAAC

tig-RT-R：CTTCAACTTTCTTCAGGTTG

2. Construction of BL21 (DE 3) ΔlpxM tig ⁺ Mutant strains

The tig-cassette expression cassette comprising the T7 promoter and terminator was amplified using the pET28a-tig vector as a template. The genomic DNA extracted from BL21 (DE 3) DeltalpxM mutant was used as template, left and right arms of lpxM gene were amplified, and left and right arms of lpxM were connected to both ends of tig-cassette fragment by overlap PCR, and the recombined fragment was electrically transformed into BL21 (DE 3) DeltalpxM/pCas competent cells together with pTargetF-sgRNA1 and pTargetF-sgRNA2 vector. The genomic DNA of the transformants was extracted as a template for PCR amplification, and the genomic DNA of BL21 (DE 3) and BL21 (DE 3) ΔlpxM were used as controls. As a result, the size of the fragment amplified by taking the genomic DNA extracted from the positive transformant as a template is about 2750bp, which is about 1200bp larger than the band amplified by taking the BL21 (DE 3) genomic DNA as a template, which is about 1800bp larger than the band amplified by taking the BL21 (DE 3) DeltalpxM mutant genomic DNA as a template. Semi-quantitative analysis is carried out on the transcription level of the tig gene by RT-PCR, and the result shows that the positive transformant can amplify a target band, and the success of the integration and expression of the tig gene is verified.

3. Construction of recombinant vector pET28a-Rcodon

1. Design of synthetic primers

The gene sequence containing tRNA elements (shown as SEQ. ID NO 1) was synthesized into pUC57 vector by Suzhou Jin Weizhi Biotechnology Co., ltd. And primer design was performed based on the commercial vector pET28a and the synthesized pUC57-Rcodon vector gene sequence.

Rcodon-F：TAAGGCAGTTATTGGTGCCCTTAAACGCCT

Rcodon-R：ATCATCTTATTAATCAGATAAAATATTTCT

pETbone-F：

AGAAATATTTTATCTGATTAATAAGATGATCAGCTTGTCTGTAAGCGGAT

pETbone-R：

AGGCGTTTAAGGGCACCAATAACTGCCTTATCAAGGGCATCGGTCGAGAT

2. Construction of pET28a-Rcodon recombinant vector

Amplifying skeleton parts except for ori and Lac I element intervals by taking pET28a as a template, wherein the size of the skeleton parts is about 4300 bp; the pUC57-Rcodon is used as a template to amplify a sequence fragment containing seven tRNA coding genes, the size of the fragment is about 2800bp, and two ends of the fragment respectively have 20bp motifs homologous to a pET28a skeleton. UsingUni Seamless Cloning and Assembly Kit the backbone fragment of pET28a is recombined with a fragment containing the tRNA encoding gene, and the transformant is screened after the DH5 alpha competent cells are transformed by the recombination system. Transformant plasmids were extracted and sequenced by Souzhou Jin Weizhi Biotechnology Co., ltd, and the recombinant vector (pET 28 a-Rcodon) was verified to be correct.

4. Expression of IBDV VP2 protein

1. Design of synthetic primers

The sequence of the IBDV VP2 gene in GeneBank (Gene accession number: KF 021490.1) was designed by itself using Primer5.0.

VP2-F：GGGAATTCCATATGATGACCAACCTGCAGGACCA

VP2-R：CCGCTCGAGTTAACGCAGAGCACGGATGA

2. Cloning VP2 gene sequence to pET28a-Rcodon recombinant expression vector

The VP2 sequence of IBDV and pET28a-Rcodon recombinant vector are subjected to double digestion by using Nde I and Xho I, and DH5 alpha competent cells are transformed after digestion product connection. After extracting transformant plasmid, double enzyme digestion verification is carried out by Nde I and Xho I, target fragments with the sizes of about 7100bp and 1359bp can be obtained, and the construction success of recombinant vector pET28a-Rcodon-VP2 is verified. The VP2 sequence is cloned into a pET28a vector according to the same method, and double digestion verification is carried out through Nde I and Xho I, so that target fragments with the sizes of 5369bp and 1359bp are obtained.

3. Expression of VP2 protein

Recombinant vector pET28a-Rcodon-VP2 converts BL21 (DE 3)ΔlpxM tig ⁺ The strain, pET28a-VP2 vector, was transformed into BL21 (DE 3) strain and inoculated into LB medium containing 50-100. Mu.g/ml kanamycin in an amount of 1% (V/V), and cultured with shaking at 37 ℃. When od600=0.4 to 0.6, the mixture was left at 28 ℃ for 30 minutes. isopropyl-beta-D-thiopyran galactoside (IPTG) is added to make the final concentration of the mixture be 0.1-1.0 mM, and the mixture is cultured for 24 hours at 28 ℃ in an oscillating way. After the completion of the culture, the cells were collected, resuspended in PBS (sodium chloride, 8g, potassium chloride, 0.2g, disodium hydrogen phosphate, 1.44g, potassium dihydrogen phosphate, 0.24g, pH 7.4, constant volume 1L), sonicated, and centrifuged to obtain the supernatant. Results BL21 (DE 3) ΔlpxM tig ⁺ The endotoxin level of the strain broken supernatant is obviously reduced compared with BL21 (DE 3) strain, and the endotoxin levels of the strain broken supernatant and the strain are respectively 5 multiplied by 10 ⁴ Eu/mL and 5X 10 ⁵ Eu/mL. SDS-PAGE results show that VP2 protein exists in BL21 (DE 3) strain in the form of inclusion body in a large part, and is expressed in a soluble way in a part. And at BL21 (DE 3) ΔlpxM tig ⁺ In the strain, the soluble expression level of VP2 protein is obviously improved.

Example 2 preparation of infectious bursal disease Virus VP2 protein subunit vaccine

The chicken infectious bursal disease virus VP2 proteins expressed by the different hosts of example 1 were mixed with mineral oil adjuvant in a ratio, and 1% thiomersal solution was added to a final concentration of 0.01% before stopping stirring. The specific proportions are shown in Table 1.

TABLE 1 chicken infectious bursal disease Virus VP2 protein subunit vaccine formulation

Component (A)	Vaccine 1	Vaccine 2
			pET28a-Rcodon-VP2 antigen (AGP potency)	1:8	-
pET28a-VP2 antigen (AGP potency)	-	1:8
			Mineral oil adjuvant (V/V%)	66％	66％

Example 3 safety test of infectious bursal disease Virus VP2 protein subunit vaccine

30 SPF chickens of 21 days old were taken and divided into 3 groups of 10, and each of the 1 st to 2 nd groups was subcutaneously immunized with vaccine 1 and vaccine 2 prepared in example 2 via the neck, respectively, at an immunization dose of 0.25ml, and the 3 rd group was subcutaneously immunized with 0.25ml of physiological saline as a blank. The immune parts of all individuals after the injection of the vaccine 2 show slight muscle congestion, edema and induration, and have slight diarrhea, listlessness, anorexia and other side reaction symptoms, and all individuals after the injection of the vaccine 1 and blank control groups have no obvious clinical symptoms. The results are shown in Table 2.

TABLE 2 safety test results of chicken infectious bursal disease Virus VP2 protein subunit vaccine

The method shows that the toxicity level of the exogenous protein endotoxin expressed by the escherichia coli constructed by the method is obviously reduced, and the prepared subunit vaccine has higher safety.

Example 4 preparation of African swine fever Virus CD2v protein

Referring to the method of example 1, an E.coli strain which efficiently expresses the African swine fever virus CD2v protein is constructed, and the gene sequence of the African swine fever virus CD2v protein is shown as SEQ ID NO 2.

The constructed E.coli strain was inoculated into LB medium containing 50-100. Mu.g/ml kanamycin in an amount of 1% (V/V), and cultured with shaking at 37 ℃. When od600=0.4 to 0.6, the mixture was left at 28 ℃ for 30 minutes. isopropyl-beta-D-thiopyran galactoside (IPTG) is added to make the final concentration of the mixture be 0.1-1.0 mM, and the mixture is cultured for 24 hours at 28 ℃ in an oscillating way. After the completion of the culture, the cells were collected, resuspended in PBS (sodium chloride, 8g, potassium chloride, 0.2g, disodium hydrogen phosphate, 1.44g, potassium dihydrogen phosphate, 0.24g, pH 7.4, constant volume 1L), sonicated, and centrifuged to obtain the supernatant. Endotoxin level of 4X 10 by detection of kit ⁴ Eu/mL. SDS-PAGE results show that the soluble expression level of the CD2v protein is obviously improved, and protein quantification is carried out by referring to a BCA protein concentration determination kit method of Biyundian corporation, and the protein content is 0.4g/L.

Example 5 preparation of porcine pseudorabies virus gD protein subunit vaccine

Referring to the method of example 1, an escherichia coli strain for efficiently expressing porcine pseudorabies virus gD protein is constructed, and the gene sequence of the porcine pseudorabies virus gD protein is shown as SEQ ID NO 3.

The constructed E.coli strain was inoculated into LB medium containing 50-100. Mu.g/ml kanamycin in an amount of 1% (V/V), and cultured with shaking at 37 ℃. When od600=0.4 to 0.6, the mixture was left at 28 ℃ for 30 minutes. isopropyl-beta-D-thiopyran galactoside (IPTG) is added to make the final concentration of the mixture be 0.1-1.0 mM, and the mixture is cultured for 24 hours at 28 ℃ in an oscillating way. After the completion of the culture, the cells were collected, resuspended in PBS (sodium chloride, 8g, potassium chloride, 0.2g, disodium hydrogen phosphate, 1.44g, potassium dihydrogen phosphate, 0.24g, pH 7.4, constant volume 1L), sonicated, and centrifuged to obtain the supernatant. Endotoxin level of 4X 10 by detection of kit ⁴ Eu/mL. SDS-PAGE results show that the soluble expression level of the gD protein is obviously improved, and protein quantification is carried out by referring to a BCA protein concentration determination kit method of Biyundian corporation, and the protein content is 3g/L.

Uniformly mixing the porcine pseudorabies virus gD protein expressed by the escherichia coli with 206 adjuvants according to a proportion, stirring for 15 minutes at 30 ℃ at 120 r/min, and preserving at 4 ℃ to obtain the porcine pseudorabies virus gD protein subunit vaccine composition. The specific proportions are shown in Table 3.

Table 3 PRRSV gD protein subunit vaccine formulation

Component (A)	Vaccine 3	Vaccine 4
			gD protein (μg/ml)	20	100
206 adjuvant (V/V%)	46	46

Example 6 porcine pseudorabies virus gD protein subunit vaccine immunogenicity test

The 21-day-old PRV antigen antibody-negative piglets were randomly divided into 3 groups, 4 groups/group, 4 groups, 5 groups were respectively injected with the vaccine 3 and the vaccine 4 prepared in example 5 by muscle, the immunization dose was 2 ml/head, and the 6 group control group was injected with 2 ml/head of DMEM medium. 28 days after immunization, the porcine pseudorabies virus HN1201 strain (Pseudorabies virus, strain HN 1201) with a preservation number of CCTCC No. V201311, a preservation unit of China center for type culture Collection, a preservation address of university of Wuhan and Wuhan, a preservation date of 2013, 5 months and 20 days, disclosed in Chinese patent application CN 104004774A) is used for treating viral fluid by intramuscular injection, and the dosage is 2×10 ^8.0 TCID ₅₀ After toxin is removed, the body temperature of the piglet is measured every day, and clinical symptoms and death conditions are observed. The results are shown in Table 4.

TABLE 4 results of porcine pseudorabies virus gD protein subunit vaccine immunogenicity test

Group of	Clinical symptoms and death conditions	Protection rate
			4	The body temperature is raised for 1-2 days, appetite is normal, no mental symptoms exist basically, and survival is realized	100％(4/4)
5	The body temperature is raised for 1-2 days, appetite is normal, no mental symptoms exist basically, and survival is realized	100％(4/4)
			6	Has obvious symptoms, 2 days after toxin attack, 2 deaths and 3 days of total deaths	0％(0/4)

The result shows that 100% (4/4) protection can be provided for piglets after the piglets are immunized by the porcine pseudorabies virus gD protein subunit vaccine, and the control piglets die all after 4 days after virus attack, so that good immune protection is shown.

Example 7 preparation of avian adenovirus Fiber-2 protein subunit vaccine

Referring to the method of example 1, an E.coli strain which efficiently expresses the avian adenovirus Fiber-2 protein is constructed, and the gene sequence of the avian adenovirus Fiber-2 protein is shown as SEQ ID NO 4.

The constructed E.coli strain was inoculated into LB medium containing 50-100. Mu.g/ml kanamycin in an amount of 1% (V/V), and cultured with shaking at 37 ℃. When od600=0.4 to 0.6, the mixture was left at 28 ℃ for 30 minutes. isopropyl-beta-D-thiopyran galactoside (IPTG) is added to make the final concentration of the mixture be 0.1-1.0 mM, and the mixture is cultured for 24 hours at 28 ℃ in an oscillating way. After the completion of the culture, the cells were collected, resuspended in PBS (sodium chloride, 8g, potassium chloride, 0.2g, disodium hydrogen phosphate, 1.44g, potassium dihydrogen phosphate, 0.24g, pH 7.4, constant volume 1L), sonicated, and centrifuged to obtain the supernatant. Endotoxin level of 4X 10 by detection of kit ⁴ Eu/mL. SDS-PAGE results show that the soluble expression level of the Fiber-2 protein is obviously improved, the content of the target protein is higher, and the AGP titer of the Fiber-2 protein reaches 1:128.

The avian adenovirus Fiber-2 protein expressed by the escherichia coli and the mineral oil adjuvant are mixed uniformly according to a proportion, and 1 percent of merthiolate solution is added before stirring is stopped, so that the final concentration is 0.01 percent. The specific proportions are shown in Table 5.

TABLE 5 avian adenovirus Fiber-2 protein subunit vaccine formulation

Component (A)	Vaccine 5	Vaccine 6
			Fiber-2 protein (AGP potency)	1:4	1:32
Mineral oil adjuvant (V/V%)	66％	66％

Example 8 avian adenovirus Fiber-2 protein subunit vaccine immunogenicity test

30 SPF chickens of 21 days old are divided into 3 groups of 10 SPF chickens, the vaccine 5 and the vaccine 6 prepared in the immunization example 7 are respectively injected into the neck by subcutaneous injection, the immunization dose is 0.3ml, and the physiological saline is 0.3ml by subcutaneous injection in the 9 th group to be used as a virus attack control. All tested chickens were kept separately and were challenged with FAV-HN strain (Fowl aviadenovirus, strain FAV-HN) at accession No. CCTCC No. V201609, china center for type culture Collection at accession No. Wuhan university, china for a period of 29 days at 2016, 2 months, and published in China patent application CN 107523556A) for 14 days by intramuscular injection, and the numbers of morbidity, mortality and protection were recorded. The results are shown in Table 6.

TABLE 6 results of avian adenovirus Fiber-2 protein subunit vaccine immunogenicity test

The results show that the 9 th group of the virus-attack control group is all ill and dead, and the 7 th to 8 th immune groups generate better immune protection and good immune effect on immunized chickens. The avian adenovirus Fiber-2 protein subunit vaccine prepared by the method can provide effective immune protection for chicken flocks.

Example 9 preparation of avian egg drop syndrome Virus tFiber protein subunit vaccine

Referring to the method of example 1, an E.coli strain is constructed which efficiently expresses the tFiber protein of the avian egg drop syndrome virus, the gene sequence of which is shown in SEQ ID NO 5.

The constructed E.coli strain was inoculated into LB medium containing 50-100. Mu.g/ml kanamycin in an amount of 1% (V/V), and cultured with shaking at 37 ℃. When od600=0.4 to 0.6, the mixture was left at 28 ℃ for 30 minutes. Adding isopropyl groupbeta-D-thiogalactopyranoside (IPTG) was cultured at a final concentration of 0.1 to 1.0mM with shaking at 28℃for 24 hours. After the completion of the culture, the cells were collected, resuspended in PBS (sodium chloride, 8g, potassium chloride, 0.2g, disodium hydrogen phosphate, 1.44g, potassium dihydrogen phosphate, 0.24g, pH 7.4, constant volume 1L), sonicated, and centrifuged to obtain the supernatant. Endotoxin level of 3X 10 by detection of kit ⁴ Eu/mL. SDS-PAGE results show that the soluble expression level of tFiber protein is obviously improved, the content of target protein is higher, and the AGP titer of the tFiber protein reaches 1:512.

The fowl egg drop syndrome virus tFiber protein expressed by colibacillus and mineral oil adjuvant are uniformly mixed according to a certain proportion, and 1% thiomersal solution is added before stopping stirring so as to make its final concentration be 0.01%. The specific proportions are shown in Table 7.

TABLE 7 avian egg drop syndrome Virus tFiber protein subunit vaccine formulation

Component (A)	Vaccine 7	Vaccine 8
			tFiber protein (AGP potency)	1:8	1:64
Mineral oil adjuvant (V/V%)	66％	66％

Example 10 avian egg drop syndrome Virus tFiber protein subunit vaccine immunogenicity test

30 SPF chickens of 21 days old were divided into 3 groups of 10, and 10 to 11 groups were subcutaneously immunized with vaccine 7 and vaccine 8 prepared in example 9 by neck, respectively, at an immunization dose of 0.5ml, and 12 groups were subcutaneously immunized with 0.5ml of physiological saline as a blank. All test chickens were kept separately and each chicken was bled separately 21 days after immunization, serum was isolated and the serum avian egg drop syndrome HI antibody titers were determined. The results are shown in Table 8.

TABLE 8 results of avian egg drop syndrome Virus tFiber protein subunit vaccine immunogenicity test

The results show that the HI antibody titer of the 12 th control group after immunization is 0 on 21 days, and the 10 th to 11 th immune groups generate higher HI antibody titers on immunized chickens, so that the immune effect is good. The poultry egg drop syndrome virus tFiber protein subunit vaccine prepared by the method can provide effective immune protection for chicken flocks.

EXAMPLE 11 preparation of the VP60 protein of Rabbit pestivirus

Referring to the method of example 1, an E.coli strain which efficiently expresses the VP60 protein of the rabbit pestivirus is constructed, and the gene sequence of the VP60 protein of the rabbit pestivirus is shown as SEQ ID NO 6.

The constructed E.coli strain was inoculated into LB medium containing 50-100. Mu.g/ml kanamycin in an amount of 1% (V/V), and cultured with shaking at 37 ℃. When od600=0.4 to 0.6, the mixture was left at 28 ℃ for 30 minutes. isopropyl-beta-D-thiopyran galactoside (IPTG) is added to make the final concentration of the mixture be 0.1-1.0 mM, and the mixture is cultured for 24 hours at 28 ℃ in an oscillating way. After the completion of the culture, the cells were collected, resuspended in PBS (sodium chloride, 8g, potassium chloride, 0.2g, disodium hydrogen phosphate, 1.44g, potassium dihydrogen phosphate, 0.24g, pH 7.4, constant volume 1L), sonicated, and centrifuged to obtain the supernatant. Endotoxin level of 4X 10 by detection of kit ⁴ Eu/mL. SDS-PAGE results show that the soluble expression level of VP60 protein is obviously improved, the content of target protein is higher, and the HA titer of VP60 protein reaches 16log2.

Example 12 preparation of porcine circovirus 3 Cap protein subunit vaccine

Referring to the method of example 1, an E.coli strain which efficiently expresses porcine circovirus type 3 Cap protein is constructed, and the gene sequence of the porcine circovirus type 3 Cap protein is shown as SEQ ID NO 7.

The constructed E.coli strain was inoculated into LB medium containing 50-100. Mu.g/ml kanamycin in an amount of 1% (V/V), and cultured with shaking at 37 ℃. When od600=0.4 to 0.6, the mixture was left at 28 ℃ for 30 minutes. isopropyl-beta-D-thiopyran galactoside (IPTG) is added to make the final concentration of the mixture be 0.1-1.0 mM, and the mixture is cultured for 24 hours at 28 ℃ in an oscillating way. After the completion of the culture, the cells were collected, resuspended in PBS (sodium chloride, 8g, potassium chloride, 0.2g, disodium hydrogen phosphate, 1.44g, potassium dihydrogen phosphate, 0.24g, pH 7.4, constant volume 1L), sonicated, and centrifuged to obtain the supernatant. Endotoxin level of 5X 10 by detection of kit ⁴ Eu/mL. SDS-PAGE results show that the soluble expression level of the porcine circovirus type 3 Cap protein is obviously improved, and protein quantification is carried out by referring to a BCA protein concentration determination kit method of Biyundian corporation, and the protein content is 0.5g/L.

Uniformly mixing the porcine circovirus 3 type Cap protein expressed by the escherichia coli and a water-soluble adjuvant Gel adjuvant (French Sibirch Co.) according to a proportion to obtain the porcine circovirus 3 type Cap protein subunit vaccine composition. The specific proportions are shown in Table 9.

Table 9 pig circovirus 3 Cap protein subunit vaccine formulation

Component (A)	Vaccine 9	Vaccine 10
			Cap protein (μg/ml)	25	100
Gel adjuvant (V/V%)	10％	10％

Example 13 porcine circovirus 3 Cap protein subunit vaccine immunogenicity test

Healthy piglets of which the ages of 28-30 days are subjected to ELISA detection on PCV2, PCV3 antigens and antibody negative are randomly divided into 3 groups and 5 groups, and the vaccine of the porcine circovirus 3 Cap protein subunit prepared in immunization example 12 is prepared. The 13 th to 14 th groups are immunized with 9 to 10 vaccine respectively, and the 15 th group is not immunized as a virus-counteracting control group. Each immunization group was injected with 2 ml/head of vaccine, and the control group was inoculated with 2 ml/head of physiological saline. The virus is removed 28 days after immunization, the dose of the virus is SG strain porcine circovirus (porcine circovirus 3 SG strain (Porcine Circovirus type, strain SG) is preserved in China center for type culture Collection (CCTCC NO. V201712), the preservation date is 23 days of 2017, and the preservation address is that of university of Wuhan and Wuhan in China, disclosed in China patent application CN 108660115A) 10 ^5.0 TCID ₅₀ And (3) continuously observing each piglet after the virus attack, and judging according to the clinical symptoms, pathological changes and virus detection results of each piglet, wherein the specific results are shown in Table 10.

TABLE 10 results of porcine circovirus 3 Cap protein subunit vaccine immunogenicity test

The result shows that after the pig circovirus 3 Cap protein subunit vaccine is used for immunizing piglets, 100% (5/5) protection can be provided for the piglets, and the control piglets are all ill after virus attack. The porcine circovirus 3 Cap protein subunit vaccine prepared by the method can provide effective immune protection for a pig group.

EXAMPLE 14 preparation of porcine circovirus 2 Cap protein

Referring to the method of example 1, an E.coli strain is constructed which efficiently expresses porcine circovirus type 2 Cap protein, and the porcine circovirus type 2 Cap gene sequence is disclosed in Chinese patent application CN101920012A.

The constructed E.coli strain was inoculated into LB medium containing 50-100. Mu.g/ml kanamycin in an amount of 1% (V/V), and cultured with shaking at 37 ℃. When od600=0.4 to 0.6, the mixture was left at 28 ℃ for 30 minutes. isopropyl-beta-D-thiopyran galactoside (IPTG) is added to make the final concentration of the mixture be 0.1-1.0 mM, and the mixture is cultured for 24 hours at 28 ℃ in an oscillating way. After the completion of the culture, the cells were collected, resuspended in PBS (sodium chloride, 8g, potassium chloride, 0.2g, disodium hydrogen phosphate, 1.44g, potassium dihydrogen phosphate, 0.24g, pH 7.4, constant volume 1L), sonicated, and centrifuged to obtain the supernatant. Endotoxin level of 5X 10 by detection of kit ⁴ Eu/mL. SDS-PAGE results show that the soluble expression level of Cap protein is obviously improved, and protein quantification is carried out by referring to a BCA protein concentration determination kit method of Biyundian corporation, and the protein content is 0.6g/L.

Example 15 preparation of porcine parvovirus VP2 protein

Referring to the method of example 1, an E.coli strain is constructed which efficiently expresses porcine parvovirus VP2 protein, and the gene sequence of porcine parvovirus VP2 protein is disclosed in Chinese patent application CN103908664A.

The constructed E.coli strain was inoculated into LB medium containing 50-100. Mu.g/ml kanamycin in an amount of 1% (V/V), and cultured with shaking at 37 ℃. When od600=0.4 to 0.6, the mixture was left at 28 ℃ for 30 minutes. isopropyl-beta-D-thiopyran galactoside (IPTG) is added to make the final concentration of the mixture be 0.1-1.0 mM, and the mixture is cultured for 24 hours at 28 ℃ in an oscillating way. After the completion of the culture, the cells were collected and washed with PBS (sodium chloride, 8g, potassium chloride, 0.2g, dibasic phosphate)Sodium, 1.44g, potassium dihydrogen phosphate, 0.24g, pH of 7.4, constant volume of 1L), and centrifuging to obtain supernatant. Endotoxin level of 4X 10 by detection of kit ⁴ Eu/mL. SDS-PAGE results show that the soluble expression level of VP2 protein is obviously improved, and protein quantification is carried out by referring to a BCA protein concentration determination kit method of Biyundian corporation, and the protein content is 2.1g/L.

EXAMPLE 16 preparation of Swine fever Virus E2 protein

Referring to the method of example 1, an E.coli strain was constructed which efficiently expressed the E2 protein of swine fever virus, and the E2 protein gene sequence of swine fever virus was disclosed in Chinese patent application CN105527442A.

The constructed E.coli strain was inoculated into LB medium containing 50-100. Mu.g/ml kanamycin in an amount of 1% (V/V), and cultured with shaking at 37 ℃. When od600=0.4 to 0.6, the mixture was left at 28 ℃ for 30 minutes. isopropyl-beta-D-thiopyran galactoside (IPTG) is added to make the final concentration of the mixture be 0.1-1.0 mM, and the mixture is cultured for 24 hours at 28 ℃ in an oscillating way. After the completion of the culture, the cells were collected, resuspended in PBS (sodium chloride, 8g, potassium chloride, 0.2g, disodium hydrogen phosphate, 1.44g, potassium dihydrogen phosphate, 0.24g, pH 7.4, constant volume 1L), sonicated, and centrifuged to obtain the supernatant. Endotoxin level of 4X 10 by detection of kit ⁴ Eu/mL. SDS-PAGE results show that the soluble expression level of E2 protein is obviously improved, and protein quantification is carried out by referring to a BCA protein concentration determination kit method of Biyundian corporation, and the protein content is 2.8g/L.

EXAMPLE 17 preparation of the GALE protein of Schistosoma japonicum

Referring to the method of example 1, an E.coli strain was constructed which efficiently expressed the GALE protein of Schistosoma japonicum, and the GALE protein gene sequence of Schistosoma japonicum was disclosed in Chinese patent application CN102079783A.

The constructed E.coli strain was inoculated into LB medium containing 50-100. Mu.g/ml kanamycin in an amount of 1% (V/V), and cultured with shaking at 37 ℃. When od600=0.4 to 0.6, the mixture was left at 28 ℃ for 30 minutes. isopropyl-beta-D-thiopyran galactoside (IPTG) is added to make the final concentration of the mixture be 0.1-1.0 mM, and the mixture is cultured for 24 hours at 28 ℃ in an oscillating way. After the culture is finished, the thalli are collected and PBS is used forSodium chloride, 8g, potassium chloride, 0.2g, disodium hydrogen phosphate, 1.44g, monopotassium phosphate, 0.24g, pH value of 7.4 and constant volume of 1L) to resuspend the thallus, ultrasonic crushing and centrifuging to obtain the supernatant. Endotoxin level of 5X 10 by detection of kit ⁴ Eu/mL. SDS-PAGE results show that the soluble expression level of GALE protein is obviously improved, and protein quantification is carried out by referring to a BCA protein concentration determination kit method of Biyundian corporation, and the protein content is 1.3g/L.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

Sequence listing

<110> Protect bioengineering Co., ltd

<120> a method for efficiently expressing exogenous protein in E.coli expression system and application thereof

<130> 19NAP0459C

<160> 7

<170> PatentIn version 3.5

<210> 1

<211> 2850

<212> DNA

<213> artificial sequence

<400> 1

taaggcagtt attggtgccc ttaaacgcct ggttgctacg cctgaataag tgataataag 60

cggatgaatg gcagaaattc gaaagcagat tcgacccggt cgtcggttca gggcagggtc 120

gttaaatagc cgcttatgtc tattgctggt ttaccggttt attgactacc ggaagcagtg 180

tgaccgtgtg cttctcaaat gcctgaggtc tgtttatcga attaattgca gatataaaaa 240

aaccaaccgt aagggttggt tttttcttgg gatttttggt cggcacgaga ggatttgaac 300

ctccgacccc cgacacccca tgacggtgcg ctaccaggct gcgctacgtg ccgactcgtg 360

gctgctaata ctaccgtttt ccacaccgat tgcaagtaag atatttcgct aactgattta 420

taattaatca gttagcgata aaacgcttct cgtacaacgc tttctggtga atggtgcggg 480

aggcgagact tgaactcgca caccttgcgg cgccagaacc taaatctggt gcgtctacca 540

atttcgccac tcccgcaaaa aaagatggtg gctacgacgg gattcgaacc tgtgacccca 600

tcattatgag tgatgtgctc taaccaactg agctacgtag ccatcttttt tttcgcgata 660

ccttatcggc gttgcggggc gcattatgcg tatagagcct tgcagcgtca acctcttttt 720

caaggaaaat tgctcgaaag tgactgtttg gttaggttgc gaacagcgaa ccatgacgaa 780

ctgtaaatct acggaatgct tgatattcag gggatttttg cggactggta cggatgggag 840

cgaactgata aatggtgtcc cctgcaggaa tcgaacctgc aattagccct taggaggggc 900

tcgttatatc catttaacta agaggacaat gcggcatgag tatacccgct aatggagtgc 960

ggggtaagta cgttgccgct cgattgctta aaccctcgcc atttatgccg ggtttttata 1020

atttttctta atgttttccg cacgttctgc tttttggacg tcatcgattg tccctctaag 1080

acacggataa atcggtgata tcaccacatc aaccaggcaa catgcccgac ttgttgaatg 1140

caataaacag aaggaaaaaa cagggaggag aaaagaagtg gtgctgatag gcagattcga 1200

actgccgacc tcacccttac caagggtgcg ctctaccaac tgagctatat cagcacatct 1260

tggagcgggc agcgggaatc gaacccgcat catcagcttg gaaggctgag gtaatagcca 1320

ttatacgatg cccgcatcct ggaactcggc tacctgattt tcattctgca ctgaatatcg 1380

agagaagctc tctttattcg agccggtaag cgaacttatc gtctcgggct acgccatcgc 1440

gtggccgaaa ttggtggtgg gggaaggatt cgaaccttcg aagtctgtga cggcagattt 1500

acagtctgct ccctttggcc gctcgggaac cccaccggac ttgatggtgc cgactaccgg 1560

aatcgaactg gtgacctact gattacaagt cagttgctct acctactgag ccaagtcggc 1620

atcaagtagc gcgcactcta tggagacatg cgagttcatg caactaaaaa attgcataat 1680

ttgttttatt ggtcacattt tatgcgacac gatgaagaaa cagcctaggt acctcatgag 1740

cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac 1800

cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga tccgtcacat 1860

accaaggcgg ctaagcgagc agatggaaca tcaacgcctg cggtcaggaa gatgcgcatc 1920

gacagcaaga catcatggct atcagcttgt cgtcgtgcag gaattgaaga tttccgtttc 1980

catgacctca gacacacctg ggcaagctgg ctgattcagt caggcgtccc attatcagtg 2040

cttcaggaaa tgggcggatg ggagtccata gaaatggttc gtaggtatgc tcaccttgcg 2100

cctaatcatt tgacagagca tgcgaggaaa atagacgaca tttttggtga taatgtccca 2160

aatatgtccc actctgaaat tatggaggat ataaagaagg cgtaactgat tgaattgtaa 2220

tggcgcgccc tgcaggattc gaacctgcgg cccacgactt agaaggtcgt tgctctatcc 2280

aactgagcta agggcgcgtt gataccgcaa tgcggtgtaa tcgcgtgaat tatacggtca 2340

acccttgctg agtcaatggc ttttgatctg gttgctgaac aagtgaacga ccgcgtctga 2400

ttttctgatt tatttcgcta tagcggcaaa caaacgcaca ccgctgcgcg tctgaatcaa 2460

gaaaacccgt attttcatgt atcaaagtga cctgcagcca agcttggatt gcgacacgga 2520

gttactttat aatccaatcg attggcccct tagctcagtg gttagagcag gcgactcata 2580

atcgcttggt cgctgcttca agtccagcag gggccaccag cggccgcaaa ggctgacgag 2640

aaatcgtcag cctttttaag ctttaagccg aattccagca cactggcggc cgttactagt 2700

ggatccgagc tcggtaccaa gcttatcgat gataagctgt caaacatgag aattacaact 2760

tatatcgtat ggggctgact tcaggtgcta catttgaaga gataaattgc actgaaatct 2820

agaaatattt tatctgatta ataagatgat 2850

<210> 2

<211> 498

<212> DNA

<213> African swine fever virus

<400> 2

ggtgtttctt ggaacttctt caacaactct ttcaacaccc tggctacctg cggtaaagct 60

ggtaacttct gcgaatgctc taactactct acctctatct acaacatcac caacaactgc 120

tctctgacca tcttcccgca caacgacgtt ttcgacacca cctaccaggt tgtttggaac 180

cagatcatca actacaccat caaactgctg accccggcta ccccgccgaa catcacctac 240

aactgcacca acttcctgat cacctgcaaa aaaaacaacg gtaccaacac caacatctac 300

ctgaacatca acgacacctt cgttaaatac accaacgaat ctatcctgga atacaactgg 360

aacaactcta acatcaacaa cttcaccgct acctgcatca tcaacaacac catctctacc 420

tctaacgaaa ccaccctgat caactgcacc tacctgaccc tgtcttctaa ctacttctac 480

accttcttca aactgtac 498

<210> 3

<211> 1029

<212> DNA

<213> Pseudorabies virus

<400> 3

gctgatgtgg atgccgtgcc cgctcccacc tttcctcctc ctgcctaccc ctacaccgag 60

agctggcagc tgacactgac cacagtgcct tcccccttcg tgggccctgc cgatgtgtac 120

cacaccaggc ccctggagga tccttgcgga gtggtggctc tcatcagcga ccctcaggtc 180

gacaggctgc tgaacgaggc tgtggcccac aggaggccta catacagggc ccacgtggcc 240

tggtacagga tcgccgacgg ctgtgcccac ctgctgtact ttatcgagta cgctgactgc 300

gaccccaggc agattttcgg caggtgccgg aggaggacca cccctatgtg gtggaccccc 360

tccgccgact acatgttccc caccgaggac gagctgggcc tgctgatggt ggcccctggc 420

aggttcaatg agggccagta caggaggctg gtgtccgtgg acggcgtgaa catcctcacc 480

gacttcatgg tggccctgcc tgagggccag gaatgtcctt tcgcccgggt cgaccagcac 540

cggacctaca agttcggcgc ctgctggtcc gacgactcct tcaagagggg cgtggacgtg 600

atgaggttcc tgaccccctt ctatcagcag cccccccaca gggaggtggt gaactactgg 660

tacaggaaga acggcaggac actgccccgg gcttatgctg ccgccacacc ttacgccatc 720

gaccccgcta ggcccagcgc tggatccccc aggccccgtc cccgtccccg tcctcggccc 780

cgtcctaaac ctgagcctgc ccctgctaca cctgctcccc ctggaaggct gcctgaacct 840

gctacccggg atcacgctgc tggcggaagg cctacaccca ggcctccccg tcctgagacc 900

cctcataggc ctttcgctcc ccctgctgtc gtcccttccg gatggcctca gcctgccgag 960

ccttttcccc ccaggacaac cgccgctcct ggagtctcca ggcataggca tcaccaccat 1020

caccactga 1029

<210> 4

<211> 1440

<212> DNA

<213> avian adenovirus

<400> 4

atgttacgtg ctcctaaacg gcgacatagc gaaaacggtc aaccggaatc ggaagccggc 60

cccagccctg cgccaataaa acgcgccaaa aggatggtac gagcaagcca gctagacctc 120

gtttaccctt ttgactacgt ggctgaccca gttggtggtc tcaacccccc attcctgggc 180

ggctctggtc ctctggtgga tcagggaggc cagttgaccc taaatgttac ggaccctatc 240

ataataaaga atcgctccgt ggacctggca catgacccct ctctagacgt caatgcccag 300

ggacaactag cagttgctgt tgaccctgag ggtgctttgg acatcacacc agatggccta 360

gatgtgaagg tagatggggt caccgtgatg gtgaatgatg actgggaact ggctgtgaag 420

gtggacccca gcggtgggtt ggacagtaca gcagggggcc tgggggtgtc tgtcgatgat 480

actttactcg tggaccaggg cgaactggga gtgcacctga accagcaagg acctatcacc 540

gcagactcca gcgggataga tctagagatc aatcccaaca tgttcaccgt caatacatcc 600

acagggagcg gtgttttaga attaaacctg aaggctcaag gtggcatcca ggctggctct 660

tcgggcgtgg gggttagtgt tgatgaatca cttgaaatcg tgaataatac actagaagtt 720

aagcctgacc cgagtggccc acttactgtg tcagccaacg gacttgggct gaaatatgat 780

tccaacactc tggccgtgac cgccggggcc ttgacggtag tgggcggcgg tagtgtctca 840

actccaattg ccacatttgt gtccggcagt ccctcgctga atacctacaa tgcgactatc 900

gtaaactcct catcccaccc cttctcctgc gcctactacc tccagcagtg gaatgtgcag 960

ggtttattat tcacatcatt atatgtgaaa ctggattcta caacaatggg cacaaggccg 1020

ggggacaaca gcagtgccaa cgccaagtgg tttactttct gggtgtccgc ctatttacaa 1080

cagtgcaacc cttccggtat tcaggccggt actgttagtc catctaccgc ggcgttagcc 1140

gatttcgaac cgatggcgaa ccggagcgta agctccccgt ggacgtactc ggctaacgcg 1200

tattatcaac ctagtagcgg ggagtttcag gttttcacgc ccgtagttac cggcgcatgg 1260

aatccgggca atatcggaat tcgtgtactg cccgtaccgg taactgcatc tggtgatcga 1320

tacacgctac tgtgttattc gttgcaatgt acgaactcgt cgatttttaa cccggcaaat 1380

agcggtacga tgatagttgg gccggttcta tactcgtgtc cggcagcctc tgtaccgtaa 1440

<210> 5

<211> 741

<212> DNA

<213> Eggdrop syndrome-1976 virus

<400> 5

ccgctgtcta tcacctctga cggtgaactg accctggctt acgactctac cgacttccag 60

gttaccgaaa acggtctggc tctgaaagtt tctccgaccc agaccccgct gacccgtatc 120

atctctatgg gtaacaacct gttcgactct ggttacgaaa tcttcgcttc ttgcccgcag 180

aacaaagctg ctaaagttgc tggttacgtt tacctgacct ctgttggtgg tctggttcac 240

ggtaccatcc agatcaaagc taccgctggt tactggttca ccggtggtaa ctctgttcag 300

gaatctatcc gtttcggtct ggttctgtgc ccgttctctg ctcgtgaccc gaccgctaac 360

ctgtctggtt ggccggctcc ggttgtttgg tctggtgact ctaacacccc gctgtacttc 420

gctgctaacg ctatctctta caccaacaac cgtgttaacc tggctgttac cggtaacttc 480

tacaaagaag aaaccgaact gccgggttac acccgtcact ctttctgccc gaccggtacc 540

accggtatga acttcaccgg tggtaacctg tacgtttgcc cgtgcaccgt taacaccggt 600

gctaccaccc tgaacgctat ctacatggtt ttcgttatca cccagtctgc tctgggtacc 660

aacttcttcg cttctaacac cccgccgaac accttcttcc tgaccccgcc gatcccgttc 720

acctacgttg gtgctcagta a 741

<210> 6

<211> 1740

<212> DNA

<213> Rabbit hemorrhagic disease virus

<400> 6

atggaaggca aagcacggac ggccccgcaa ggagaggcag ctgggactgc gacaactgca 60

tcggtccctg gaacaaccac tgatggcatg gatcctggag tcgttgccgc gacgtcagtc 120

gttaccgctg agaacagttc cgcctccgtg gcaacggcgg gcattggcgg tccaccccag 180

caggtcgacc agcaggagac atggagaaca aatttctatt acaatgatgt gttcacctgg 240

tctgtcgcag acgcgccagg tagcatcctt tacacagtgc agcacagccc tcagaacaat 300

cctttcacag ctgtgttgtc gcagatgtat gcaggctggg caggtggtat gcagtttaga 360

tttattgtag ccggaagtgg agtattcgga ggacgattag tggcagcagt tatccccccg 420

ggaattgaga tcggaccggg cctcgaagtg cgtcaattcc cacatgttgt tatcgatgca 480

agatcgttgg aacctgtaac gattactatg ccagacctgc gtccaaatat gtatcatccg 540

acaggagatc caggactagt cccgacattg gttctgagtg tgtacaacaa ccttataaac 600

ccctttggag gttctacgaa cgcgatacaa gtgaccgtgg aaacaagacc tagtgaagac 660

tttgagttcg tgatgatccg agcaccctca tcaaaaaccg tcgacagcat ttctccggct 720

ggacttctca cgacacccgt gctcacgggg gtcggcaacg acaataggtg gaacggccag 780

atcgtgggac tacaaccagt cccgggtggc ttctcgacgt gcaatagaca ctggaacctg 840

aatggctcaa catacggctg gagctcacct aggtttgcag atatagatca ccgccgtggc 900

agtgccagct attctggaaa taattcaact aatgtgctcc agttttggta tgcaaacgcc 960

ggctctgcaa ttgataaccc cattagccaa gttgcacctg atgggtttcc tgacatgtcc 1020

tttgttcctt ttaactcacc gaacatccca acagcaggct gggtgggctt tggcggcatc 1080

tggaatagta acaatggtgc ccctgctgca actactgtcc aagcatatga actgggattc 1140

gccacaggcg ctcccaacaa tctacagcct actacaaaca cttccggcgc tcagaccgtc 1200

gccaagtcta tatatgccgt cgtaactggc accaaccaga atcccacggg gctatttgta 1260

atggcgtcgg gagtcataag cacccccaat gcctcagcag ttacatatac tccacaaccc 1320

gatcgtattg ttaccacccc aggtacacct gctgcagctc cggtgggtaa gaatactcct 1380

atcatgttcg cttctgtcgt aaggagaacg ggtgatgtca atgctgcagc ggggagcacc 1440

aatgggactc agtacggtac tggtagtcag cctttaccag ttaccattgg cctctcgctg 1500

aacaactact cctctgcttt gatgcccggc cagttctttg tgtggcagct cacatttgct 1560

tcgggtttta tggagattgg tctatcggta gatggttatt tttatgccgg tacgggggca 1620

agcacaacat tgatcgatct aaccgagctc atagacgtcc gaccagtcgg tccacgcccg 1680

tcgaaaagta cacttgtttt caacttagga gggacgacaa atggtttcag ctacgtttga 1740

<210> 7

<211> 645

<212> DNA

<213> Porcine circovirus

<400> 7

atgcgtcatc gtgcgatatt tcgtcgacga ccacgaccta ggcgccggcg gcgccatcgt 60

cgccgttacg cgcggcgcaa attatttata cgtagaccga ctgcgggaac gtactacacg 120

aaaaaatatt cgacgatgaa cgtaatctcg gtaggtactc cgcaaaacaa taagccgtgg 180

cacgctaacc attttattac gcggttaaac gaatgggaga ctgcgataac tttcgaatac 240

tacaaaatat taaaaatgaa agtaactcta tctcccgtta tcagtccagc gcaacaaacg 300

aagacgatgt tcgggcatac tgcgatcgat ttggacggag cgtggactac gaatacgtgg 360

ttgcaagacg acccgtacgc cgaatcaagc acacgtaaag taatgacttc gaaaaaaaaa 420

catagccgat attttacgcc aaaaccttta ttggcgggta caacaagcgc gcacccggga 480

caatcgctat tttttttttc acgtccaacc ccgtggttga acacgtacga tccaaccgtt 540

caatggggtg ctttactttg gtcgatatac gtcccggaaa aaactggtat gactgatttt 600

tacggtacga aggaagtatg gatacgttac aaatctgtac tataa 645

Claims

1. An escherichia coli construction method for efficiently expressing exogenous proteins, wherein the method comprises the following steps:

step (1) BL21 (DE 3) lpxM gene knockout;

step (2) inserting tig genes into the BL21 (DE 3) lpxM gene deletion strain obtained in the step (1);

constructing a recombinant vector pET28a-Rcodon, wherein an Rcodon element is a gene sequence shown in SEQ ID NO.1 and comprising a tRNA element;

cloning the exogenous protein gene to the recombinant vector pET28a-Rcodon obtained in the step (3);

step (5) transforming the recombinant vector obtained in the step (4) into the strain obtained in the step (2); and

harvesting the exogenous protein in the step (6);

the exogenous proteins comprise African swine fever virus CD2v protein, avian adenovirus Fiber-2 protein, avian egg drop syndrome virus tFiber protein, chicken infectious bursal disease virus VP2 protein, porcine circovirus 3 type Cap protein, porcine circovirus 2 type Cap protein, porcine pseudorabies virus gD protein, porcine parvovirus VP2 protein, swine fever virus E2 protein, rabbit pestivirus VP60 protein and Japanese schistosome GALE protein.

2. The escherichia coli capable of efficiently expressing the foreign protein prepared by the method of claim 1.

3. A method for efficiently expressing a foreign protein, wherein the method uses the escherichia coli of claim 2 to express the foreign protein.

4. Use of the escherichia coli of claim 2 or the method of claim 3 for preparing a foreign protein.