CN1680433A - Recombinant N protein antigen and purification for transmissible gastroenteritis of swine - Google Patents

Abstract

Porcine transmissible gastroenteritis recombinant N protein antigen and its purification are disclosed. It is carried out by extracting viral nucleic acid by purified TGEV virus liquid, virogenic group cDNA synthesizing by upstream primer Li01 of TGEV N gene, using upstream and downstream primer of TGEV N gene, N gene amplifying, cloning the amplified TGEV N gene in prokaryotic expressing carrier plasmid pProEXHTb, expressing in strain of colibacillus, and obtaining porcine transmissible gastroenteritis recombinant N protein antigen. It can overcome potential risk of toxin ejection and be used for diagnostic antigen of disease monitor.

Description

Swine infectious enterogastritis recombinant N protein antigen and purification process

(1) technical field

What the present invention relates to is a kind of biotechnological formulation, the invention still further relates to the preparation method of this biotechnological formulation.Specifically a kind of swine infectious enterogastritis recombinant N protein antigen and preparation method.

(2) background technology

Transmissible gastroenteritis of swine is that (clinical symptom is serious diarrhoea, vomiting and dehydration for Transmissiblegastroenteritis virus, a kind of acute, the height contagious disease of the pig that TGEV) causes by transmissible gastro-enteritis virus.The equal susceptible of the pig of different ages and kind, especially with 2 the week ages with interior piglet case fatality rate up to 100%.

The traditional diagnosis method of transmissible gastroenteritis of swine is often according to epidemiology and the clinical manifestation foundation as tentative diagnosis.But owing to rotavirus and the Porcine epidemic diarrhea virus of pig also can cause similar disease, and on epidemiology and symptom, be difficult to difference, therefore must rely on virus separation and serology experiment to make a definite diagnosis at last.

Since nineteen forty-six Doyle and Hutchings reported first transmissible gastroenteritis of swine, big quantity research has been carried out to the TGEV diagnostic techniques in countries in the world, has set up viral separation (Isolation of Virus IV), fluorescent-antibody technique (Fluorescent Antibody TestFAT), immune electron microscopy (Immune electron microscopy IEM), virus neutralization tests (Virus Neutralization VN), enzyme linked immunosorbent assay (enzyme-linked immunosorbent assay ELISA), nucleic acid probe (Nucleic acidprod) and polymerase chain reaction methods such as (Poly Chain Reaction PCR).Can carry out tentative diagnosis to TGE according to epidemiology, clinical symptom and pathological change,, also need the laboratory to make a definite diagnosis if distinguish mutually with the intestinal tract disease that porcine rotavirus (PRV) and Porcine epidemic diarrhea virus (PEDV) cause.

Eqpidemic disease diagnosis fast and accurately is the important prerequisite of control transmissible gastroenteritis of swine.And China does not also have the effective prevention and treatment method of a cover at present.In clinical diagnosis to suspicious pathological material of disease carry out virus separate with evaluation be this disease diagnostic method the most intuitively, but isolated viral needs certain condition and considerable time, the drawback that therefore, that this diagnostic method exists is time-consuming, effort again can not quick diagnosis; This sick fast diagnosis method the most normal application be serological test, as neutralization test, fluorescent antibody test and enzyme linked immunosorbent assay etc.And the accuracy of serological method diagnosis depends on the specificity of diagnostic antigen and antibody.

(3) summary of the invention

The object of the present invention is to provide a kind ofly can overcome the existing toxin expelling of antigen in the transmissible gastroenteritis of swine traditional diagnosis method, the potentially dangerous of the poison that looses, can be used as a kind of swine infectious enterogastritis recombinant N protein antigen of diagnostic antigen of disease surveillance; The present invention also aims to provide a kind of

The antigenic purification process of swine infectious enterogastritis recombinant N protein.

The object of the present invention is achieved like this: it is to carry out viral nucleic acid (RNA) the TGEV virus liquid behind purifying to extract, upstream primer Li01 with TGEV N gene carries out the synthetic of viral genome cDNA, re-use TGEV N gene on, downstream primer, carry out the amplification of N gene, with the amplification after TGEV N gene clone in prokaryotic expression carrier plasmid pProEXHTb, and in e.colistraindh5, express and obtain swine infectious enterogastritis recombinant N protein antigen, its called after Escherichia coli DH5 a with recombinant plasmid of transmissiblegastroenteritis virus necleoprotein gene.

The upstream and downstream primer sequence is respectively:

Li01?5’AAC?TTC?GAA?ATG?GCC?AAC?C?3’ 19-mer；

Li02?5’AGC?TCG?AGC?ATC?TCG?TTT?AG?3’ 20-mer。

Swine infectious enterogastritis recombinant N protein of the present invention is to adopt such method purifying:

The great expression of 1 fusion rotein

(1) e.colistraindh5 of inoculating expressed fusion protein sways overnight incubation in 10ml LB/ penbritin liquid nutrient medium in 37 ℃;

(2) transfer the 10ml overnight culture in 500ml LB/ penbritin liquid nutrient medium, sway in 37 ℃ and be cultured to OD ₅₉₀Value reaches about 0.5, takes out 1ml sample power supply swimming and analyzes;

(3) add 0.5ml 1mol/L IPTG to final concentration be 0.6mmol/L, continue to be cultured to OD value in 37 ℃ and reach 1.0, stop cultivating, take out 1ml sample power supply swimming analysis.

The purifying of 2 fusion roteins

(1) in 1.5cm * 7.5cm chromatography column, adds 1mlNi ²⁺-NTA gel media behind the 20ml deionization washing post, is washed post with 20ml Buffer A solution again;

(2) with bacterium liquid through the centrifugal 20min of 10 000g/min, remove supernatant, sedimentary thalline carries out the cracking thalline with BufferA solution 10ml, dissolution precipitation, behind the stirring at room 1h, 4 ℃ continue to be stirred and to spend the night;

(3) solute is moved in the 30ml centrifuge tube, under 4 ℃ of conditions,, get supernatant and wait until upper prop usefulness with the centrifugal 40min of 20 000g/min;

(4) flow velocity of supernatant with 0.5ml/min joined in the Ni-NTA post; Collect effluent liquid, carry out SDS-PAGE and analyze;

(5) use 20ml Buffer A, the 20ml Buffer B flow velocity washing with 0.5ml/min successively, and collect effluent liquid, SDS-PAGE analyzes;

(6) wash with the Buffer C 10ml that contains the 80mmol/L imidazoles, collect elutriant, under the ultraviolet wavelength of 280nm, detect proteic absorption peak, preservation contains the elutriant of recombinant protein, and the storage temperature that short-term is preserved is 4 ℃, and the storage temperature of prolonged preservation is-20 ℃.

Along with the development and use of modern techniquies such as modern molecular immunology and molecular biology, for new way has been opened up in the development of biotechnology diagnostic reagent of new generation.Diagnostic antigen or antibody with advanced technique means acquisition such as genetically engineered, cell engineering, its outstanding feature is that homogeneity is good, the purity height, high specificity, stable performance, and be easy to preserve and produce in batches, diagnostor of Jian Liing and method are easy to International standardization thus, are the main directions of the development of eqpidemic disease diagnosis from now on.Simultaneously, with the diagnostic antigen that biotechnology is produced, can avoid loaded down with trivial details cell cultures to prepare a large amount of antigenic substances, this is to difficult viral extremely useful of breeding on cell.

TGEV N albumen is very conservative phosphorylated protein, and molecular mass is 47ku, be a kind of dominance structure albumen of TGEV, and can excite stronger immunne response, therefore to nucleocapsid (N) gene of TGEV clone, expression and expression product purifying thereof.No matter the nucleoprotein product of its purifying is the diagnostic antigen as a kind of disease surveillance, and still the immunological reagent of replying as a kind of excitating organism all is extremely useful novel biological agents.The further application of said preparation will provide important basic substance to the diagnosis and treatment of TGE.The diagnosis of TGE the control this disease popular on have vital role, therefore further improve these detection methods, make it to have specificity, the susceptibility of height, quicker, easy, more be applicable to the detection of conventional a large amount of samples, will have prior practical significance.

Utilize the reorganization TGEV N albumen of Protocols in Molecular Biology preparation to have the immunobiology characteristic close with natural N albumen; and can large-scale production; this technology has been avoided adopting vitro culture totivirus antigen, differential centrifugation and density gradient centrifugation purified virus, has been handled the proteic loaded down with trivial details antigen production method of exposure virus N with biochemical reagents; improve working efficiency, reduced nonspecific reaction.With the antigen that this method is produced, can avoid disseminating as antigen the danger of virus with live virus.

(4) description of drawings

Fig. 1 is the RT-PCR result of TGEV nucleoprotein gene, wherein:

1.DNA?Ladder

2-3. nucleoprotein gene PCR product

4.PCR contrast

5.DNA?Marker?DL15000

Fig. 2 is that the enzyme of TGEV N gene is cut qualification result, wherein:

1.DNA?Ladder?100bp

2.N gene is cut with Xba I enzyme

3.N gene is cut with Pst I enzyme

4.N gene is cut with the HindIII enzyme

Fig. 3 is that the enzyme of recombinant plasmid PHN is cut the product electrophoretogram, wherein:

1.DNA?Marker?DL15,000

2. vector plasmid pProEXHTb

3.XhoI single endonuclease digestion vector plasmid

4. recombinant plasmid PHN

5.XhoI single endonuclease digestion recombinant plasmid PHN

6.XhoI, NspV double digestion recombinant plasmid PHN

Fig. 4 be TGEV N gene at the SDS-PAGE of escherichia coli expression electrophoretogram, wherein:

1. standard protein molecular weight 20～90ku

2. the cellular lysate thing that contains plasmid pProEXHTb without the IPTG inductive

3. the cellular lysate thing that contains plasmid pProEXHTb through the IPTG inductive

4. the cellular lysate thing that contains recombinant plasmid PHN without the IPTG inductive

5. the cellular lysate thing that contains recombinant plasmid PHN through the IPTG inductive

Fig. 5 is the proteic Western blot of an expression in escherichia coli N collection of illustrative plates, wherein:

1, the cellular lysate thing that contains plasmid pProEXHTb without the IPTG inductive

2, the cellular lysate thing that contains plasmid pProEXHTb through the IPTG inductive

3, the cellular lysate thing that contains recombinant plasmid PHN without the IPTG inductive

4, the cellular lysate thing that contains recombinant plasmid PHN through the IPTG inductive

Fig. 6 is the electrophoretogram of fusion rotein purifying, wherein:

1, standard protein molecular weight 20～90ku

2, without IPTG inductive tropina

3, through IPTG inductive tropina

4, through buffer A cracked IPTG inductive tropina

5, through the tropina of affinitive layer purification

Fig. 7 is the fusion rotein Western-blot collection of illustrative plates behind the purifying

Fig. 8 is expression efficiency distribution of results figure

Fig. 9 is the data results table of expressing fusion protein efficiency analysis.

(5) specific embodiments

Carrying out viral nucleic acid (RNA) the TGEV virus liquid behind purifying extracts, upstream primer Li01 with TGEV N gene carries out the synthetic of viral genome cDNA, re-use TGEV N gene on, downstream primer, carry out the amplification of N gene, with the amplification after TGEV N gene clone in prokaryotic expression carrier plasmid pProEXHTb, and in e.colistraindh5, express, characteristics according to carrier, this experiment selects for use IPTG to induce Expression of Fusion Protein, expression product is after SDS-PAGE analysis and the immune marking (Western-blot) detection antigenic activity, 6 continuous histidine residues that the purifying of fusion rotein utilizes the expressed recombinant protein of prokaryotic expression carrier pProEXHT to have, can be combined on the Ni-NTA post, can be combined in Ni because of imidazoles again with competing ²⁺On-NTA the post and recombinant protein is replaced, for the extraction purifying of recombination fusion protein provides a kind of effective ways.

The concrete making method of product of the present invention is:

The extraction of TGEV TH-98 pnca gene group RNA

Proteinase K, 10%SDS cracking, add each extracting of isopyknic phenol/chloroform and chloroform once, the dehydrated alcohol precipitate nucleic acids of 2mol/L sodium acetate and 2.5 times of volumes, with 70% cold washing with alcohol precipitation, after the drying at room temperature, add an amount of (about 12 μ l) aseptic deionized water dissolving nucleic acid precipitation ,-20C preserves standby.

The synthetic the primer of RT: viral genome cDNA is the upstream primer Li01 of TGEV N gene, and reaction system and condition are undertaken by the specification sheets of ThermoScript II.The upstream and downstream primer of TGEV N gene is used in the amplification of PCR:TGEV N gene, carries out according to the specification sheets of Taq archaeal dna polymerase.

↓ RT-PCR amplification N gene and purifying

↓ XhoI, NspV double digestion N gene and vector plasmid pPRoEXHTb and purifying (purifying adopts DNA a small amount of purification kit to carry out)

Ligation is undertaken by the specification sheets of T4 dna ligase, and adds PEG 8000 to increase binding efficient; The frightened method of heat is transformed into host bacterium DH5 α.

↓ connect and be transformed into the host bacterium

Primer sequence is as follows: Li01 5 ' AAC TTC GAA ATG GCC AAC C 3 ' 19-mer; Li02 5 ' AGC TCG AGC ATC TCG TTT AG 3 ' 20-mer.

↓ positive plasmid is identified (enzyme is cut and sequencing)

↓ protein expression (IPTG induces)

↓

The extraction of plasmid is carried out with a small amount of plasmid extraction kit; Restriction enzyme XhoI and NspV carry out single endonuclease digestion and double digestion to the extraction plasmid respectively, and the product after enzyme is cut carries out check and analysis through 0.8% agarose gel electrophoresis.

SDS-PAGE and Western-blot test

↓ fusion rotein purifying

↓ Western-blot analyzes

Fig. 1 is the RT-PCR result of TGEV nucleoprotein gene, uses the RT-PCR method, and cDNA is a template with reverse transcription synthetic TGEV TH-98 strain, goes out the fragment of about 1200bp with designed primer amplification, and the amplified production size conforms to desired design (1174bp).

Fig. 2 is that the enzyme of TGEV N gene is cut qualification result, select for use restriction enzyme HindIII, Pst I, Xba I on the N gene restriction enzyme mapping that the PCR product is carried out restriction analysis, the result has obtained about 500bp and 600bp, about 350bp and 700bp and about 450bp and 700bp fragment respectively, and it is consistent with expected results that enzyme is cut qualification result.

Fig. 3 is that the enzyme of recombinant plasmid PHN is cut the product electrophoretogram, and recombinant plasmid with XhoI single endonuclease digestion and XhoI and NspV double digestion, has been obtained respectively and the fragment of estimating that size conforms to.I.e. two fragments that obtain with XhoI and the NspV double digestion 4780bp fragment that is the pProEXHTb vector plasmid and the 1174bp fragment of goal gene, and with the XhoI single endonuclease digestion clip size be sum of the two, about promptly about 6000bp.Show the recombinant plasmid that has obtained the TGEV nucleoprotein gene.

Fig. 4 be TGEV N gene at the SDS-PAGE of escherichia coli expression electrophoretogram, the result shows that reorganization cellular lysate thing produces a special protein band between 66ku and 43ku, and expects the big or small consistent of albumen (47ku).But not in the pProEXHTb plasmid bacterium culture samples of reorganization, then do not have this and take out of existing.Other is non-obvious band of expression all not occur through IPTG inductive plasmid bacterium.

Fig. 5 is a Western blot collection of illustrative plates, the DH5 α cellular lysate thing that contains recombinant plasmid PHN, after SDS-PAGE and electric transfer printing, the 47ku recombinant protein of expression can with TGEV antiserum(antisera) generation specific reaction, show the reorganization nucleoprotein have and the same antigen-specific of natural TGEV N albumen.

Fig. 6 is the electrophoretogram of fusion rotein purifying, can find out from electrophoretogram, uses this purification process finally to obtain the higher fusion rotein of purity.

Fig. 7 is the fusion rotein Western-blot collection of illustrative plates behind the purifying, and from figure as can be seen, the fusion rotein behind the purifying carries out the western-blot detection and still keeps its antigen-specific.

Fig. 8 is expression efficiency distribution of results figure, and wherein r7 is a recombinant N protein, and molecular weight is 47ku (shown in chart 9 arrows), and its content accounts for 20.409% of bacterial protein, and the result shows that fusion rotein has obtained to efficiently express in intestinal bacteria.

Sequence table

GCCAACCAGGGACAGCGTATTAGTTGGGGGGATGAATCTACCAAAACACGTGG

TCGTTCCAATTCCCGTGGTCGGAAGAATAATAACATACCTCTTTCATTCTTCAACCCC

ATAACCCTCCAACAAGGTTCAAAATTTTGGAACTTATGTCCGAGAGACTTTGTACCC

AAAGGAATAGGTAACAGGGATCAACAGATTGGTTATTGGAATAGACAAACTCGCTAT

CGCATGGTGAAGGGCCAACGTAAAGAGCTTCCTGAAAGGTGGTTCTTCTACTACTTA

GGTACTGGACCTCATGCAGATGCCAAATTTAAAGATAAATTAGATGGAGTTGTCTGG

GTTGCCAAGGATGGCGCCATGAACAAACCAACCACGCTTGGTAGTCGTGGTGCTAAT

AATGAATCCAAAGCTTTGAAATTCGATGG?TAAAGTGCCAGGCGAATTTCAACTTGAA

GTTAATCAATCAAGAGACAATTCAAGGTCACGCTCTCAATCTAGATCTCGGTCTAGA

AATAGATCTCAATCTAGAGGCAGGCAACAATTCAATAACAAGAAGGATGACAGTGTA

GAACAAGCTGTTCTTGCCGCACTTAAAAAGTTAGGTGTTGACACAGAAAAACAACA

GCAACGCTCTCGTTCTAAATCTAAAGAACGTAGTAACTCTAAGACAAGAGATACTACA

CCTAAGAATGAAAACAAACACACCTGGAAGAGAACTGCAGGTAAAGGTGATGTGAC

AAGATTTTATGGAGCTAGAAGCAGTTCAGCCAATTTTGGTGACACTGACCTCGTTGC

CAATGGGAGCAGTGCCAAGCATTACCCACAACTGGCTGAATGTGTTCCATCTGTGTC

TAGCATCTGTTTGGAAGCTATTGGACTTCAAAGGAAGATGGCGACCAGATAGAAGT

CACGTTCACACACAAATACCACTTGCCAAAGGATGATCCTAAGACTGGACAATTCCT

TCAGCAGATTAATGCCTATGCTCGTCCATCAGAAGTGGCAAAAGAACAGAGAAAAAG

AAAATCTCGTTCTAAATCTGCAGAAAGGTCAGAGCAAGATGTGGTACCTGATGCATT

AATAGAAAATTATACAGATGTGTTTGATGACACACAGGTTGAGATGATTGACGAGGT

AACGAAC

Claims (3)

1, a kind of swine infectious enterogastritis recombinant N protein antigen, it is characterized in that: it is to carry out viral nucleic acid the TGEV virus liquid behind purifying to extract, upstream primer Li01 with TGEV N gene carries out the synthetic of viral genome cDNA, re-use the upstream and downstream primer of TGEV N gene, carry out the amplification of N gene, TGEV N gene clone after the amplification in prokaryotic expression carrier plasmid pProEXHTb, and is expressed the swine infectious enterogastritis recombinant N protein antigen that obtains in e.colistraindh5.

2, swine infectious enterogastritis recombinant N protein antigen according to claim 1, it is characterized in that: gene order is:

GCCAACCAGGGACAGCGTATTAGTTGGGGGGATGA ATCTACCAAAACACGTGGTCGTTCCAATTCCCGTGGTCGGAAGAATAATAACATACCTCTTTCATTCTTCAACCCCATAACCCTCCAACAAGGTTCAAAATTTTGGAACTTATGTCCGAGAGACTTTGTACCCAAAGGAATAGGTAACAGGGATCAACAGATTGGTTATTGGAATAGACAAACTCGCTATCGCATGGTGAAGGGCCAACGTAAAGAGCTTCCTGAAAGGTGGTTCTTCTACTACTTAGGTACTGGACCTCATGCAGATGCCAAATTTAAAGATAAATTAGATGGAGTTGTCTGGGTTGCCAAGGATGGCGCCATGAACAAACCAACCACGCTTGGTAGTCGTGGTGCTAATAATGAATCCAAAGCTTTGAAATTCGATGGTAAAGTGCCAGGCGAATTTCAACTTGAAGTTAATCAATCAAGAGACAATTCAAGGTCACGCTCTCAATCTAGATCTCGGTCTAGAAATAGATCTCAATCTAGAGGCAGGCAACAATTCAATAACAAGAAGGATGACAGTGTAGAACAAGCTGTTCTTGCCGCACTTAAAAAGTTAGGTGTTGACACAGAAAAACAACAGCAACGCTCTCGTTCTAAATCTAAAGAACGTAGTAACTCTAAGACAAGAGATACTACACCTAAGAATGAAAACAAACACACCTGGAAGAGAACTGCAGGTAAAGGTGATGTGACAAGATTTTATGGAGCTAGAAGCAGTTCAGCCAATTTTGGTG ACACTGACCTCGTTGCCAATGGGAGCAGTGCCAAGCATTACCCACAACTGGCTGAATGTGTTCCATCTGTGTCTAGCATTCTGTTTGGAAGCTATTGGACTTCAAAGGAAGATGGCGACCAGATAGAAGTCACGTTCACACACAAATACCACTTGCCAAAGGATGATCCTAAGACTGGACAATTCCTTCAGCAGATTAATGCCTATGCTCGTCCATCAGAAGTGGCAAAAGAACAGAGAAAAAGAAAATCTCGTTCTAAATCTGCAGAAAGGTCAGAGCAAGATGTGGTACCTGATGCATTAATAGAAAATTATACAGATGTGTTTGATGACACACAGGTTGAGATGATTGACGAGGTAACGAAC

3, the antigenic method purifying of a kind of swine infectious enterogastritis recombinant N protein is characterized in that:

The great expression of 1 fusion rotein

(1) e.colistraindh5 of inoculating expressed fusion protein sways overnight incubation in 10ml LB/ penbritin liquid nutrient medium in 37 ℃;

(2) transfer the 10ml overnight culture in 500ml LB/ penbritin liquid nutrient medium, sway in 37 ℃ and be cultured to OD ₅₉₀Value reaches about 0.5, takes out 1ml sample power supply swimming and analyzes;

(3) add 0.5ml 1mol/L IPTG to final concentration be 0.6mmol/L, continue to be cultured to OD value in 37 ℃ and reach 1.0, stop cultivating, take out 1ml sample power supply swimming analysis.

The purifying of 2 fusion roteins

(1) in 1.5cm * 7.5cm chromatography column, adds 1ml Ni ²⁺-NTA gel media behind the 20ml deionization washing post, is washed post with 20ml Buffer A solution again;

(2) with bacterium liquid through the centrifugal 20min of 10 000g/min, remove supernatant, sedimentary thalline carries out the cracking thalline with BufferA solution 10ml, dissolution precipitation, behind the stirring at room 1h, 4 ℃ continue to be stirred and to spend the night;

(3) solute is moved in the 30ml centrifuge tube, under 4 ℃ of conditions,, get supernatant and wait until upper prop usefulness with the centrifugal 40min of 20 000g/min;

(4) flow velocity of supernatant with 0.5ml/min joined in the Ni-NTA post; Collect effluent liquid, carry out SDS-PAGE and analyze;

(5) use 20ml Buffer A, the 20ml Buffer B flow velocity washing with 0.5ml/min successively, and collect effluent liquid, SDS-PAGE analyzes;

(6) wash with the Buffer C 10ml that contains the 80mmol/L imidazoles, collect elutriant, under the ultraviolet wavelength of 280nm, detect proteic absorption peak, preservation contains the elutriant of recombinant protein, and the storage temperature that short-term is preserved is 4 ℃, and the storage temperature of prolonged preservation is-20 ℃.

Images