CN1307138A - Expression vector pBVIL1 and its construction method and use - Google Patents

Abstract

The present invention discloses expression vector with total length 4118 bp and including the great part of plasmid pBV220 and modified human IL-1 beta gene IL-1M, which contains start codon ATG, stop codon TAA and endoenzyeme sites XhoI and Xbal. The present invention also discloses the construction process of pBVIL1 and its appliication of gene cloning and expression. Owing to the low non-specific reaction of the pBVIL1 expressed fusion protein, the immunological adjuvant function of IL-IM active peptide and the cloning site of foreign gene with complementary enzyme for gene linkage, the pBVIL1 is ideal vector for antigen expression and may be applied widely.

Description

Expression vector pBVIL1 and construction process thereof and purposes

The present invention relates to the genetically engineered field, specifically expression vector pBVIL1 and construction process and purposes.

In the research of immunoassay reagent antigen, the macromole recombinant protein has non-specific responding sometimes, use small-molecular peptides can reduce non-specific responding, HCV immunity as the Ortho company third generation is that mark reagent RIBA3.0 changes the recombinant protein c22-3r of original 120 amino-acid residues into have only 44 amino acid (aa) synthetic peptide c22p, c100-3r (362aa) is changed into the c100p that has only 16aa, thereby reduced non-specific responding [Pawlotsky JM, Roudot-Thoraval F, Pellet C, Aumont P, Darthuy F, Remire J, Duval J and Dhumeaux D., Influence of hepatitis C virus (HCV) Genotypes on HCV recombinant immunoblot as say patterns, J.ClinMicrob, 1995; 35 (5): 1357-1359].Yet, in order to increase antigenic coverage rate, as ELISA reagent, sometimes need to have the fused antigen of a plurality of epitopes again, to improve sensitivity [the Chien DY that detects, Arcangel P, Medina-Selby A, Coit D, Baumeister M, Nguyen S, George-Nascimento C, Gyenes A, Kuo G, Valenzuela P.Use of a novel hepatitis C virus (HCV) major-epitope chimeric polypeptide for diagnosis of HCV infection.J Clin Microbiol 1999 May; 37 (5): 1393-7].If respectively with the synthetic little peptide of chemical synthesis, cost height not only, resultant quantity is limited, and little peptide is unfavorable for wrapping quilt, influences the sensitivity of reagent.But the ideal method is both single expression of little peptide epitope, again can be as required, a plurality of epitopes are coupled together amalgamation and expression.

PBV220 is at present domestic prokaryotic expression carrier commonly used, and [Hou Yunde contains the establishment and the application thereof of the protokaryon efficient expression vector of PRPL promotor for Zhang Zhiqing, Yao Lihong by people such as Zhang Zhiqing structure.The virus journal, 1990; 6 (2): 111-115].It contains PRPL heat shock promoter and synthetic SD sequence, Li Wuju etc. studies show that the relevant [Li Wuju of structure that reaches terminator codon sequence the last period after destination gene expression height and the initiator codon, Wu Jiajin, the quantitative analysis of external source gene expression dose in the pBV220 carrier, the virus journal, 1997; 13 (2): 126-133].

We had once made up pBV220/IL-1 β expression plasmid, made recombinant human IL-1 β obtain to express efficiently [Guo Fukun, Ling Shigan etc., IL-1 β and mutant clone thereof, expression and activity research, institute of Military Medical Science Institute periodical, 1999,23 (3): 238-9].Because people IL-1 β is a human body self protein, do not contain natural anti-people IL-1 β antibody in the human body, therefore in IL-1 β gene, select proper site to insert goal gene, under the prerequisite that does not change single open reading frame, when being used for immunologic function test reagent, the small molecules antigen of amalgamation and expression can not cause nonspecific reaction.And, fragment in people IL-1 β and the molecule thereof is very strong immunostimulant [Hakim I, Levy S, Levy R, A nine-amino acid peptidefrom IL-1 β augments antitumor immuneresponses induced byprotein and DNA vaccines.J Immunol 1996 Dec15; 157 (12): 5503-11; Beckers W, Villa L, Gonfloni S, CastagnoliL, Newton SM, Cesareni G, Ghiara P., Increasing theimmunogenicity of protein antigens through the geneticinsertion of VQGEESNDK sequence of human IL-1 β into theirsequence.J Immumol 1993 Aug 15; 151 (4): 1757-64], when expression product was used for vaccine immunogens research, the people IL-1 β fragment that merges in the molecule can play the effect of immunological adjuvant again.Based on this design, we are on the basis of pBV220/IL-1 β expression plasmid, made up a fusion expression vector pBVIL1, the small molecules Toplink has been expressed in prokaryotic cell prokaryocyte efficiently and stably, and can be realized as required interconnecting easily between the small-molecular peptides.

The purpose of this invention is to provide an efficient fusion expression carrier pBVIL1 at procaryotic cell expression.

A further object of the present invention provides the construction process of expression vector pBVIL1.

Another purpose of the present invention provides the purposes of expression vector pBVIL1 pBVIL1 in gene clone, antigen presentation and vaccine immunogens preparation.

According to a first aspect of the invention, pBVIL1 carrier disclosed by the invention is that total length is the closed loop plasmid of 4118bp as shown in Figure 1.Described carrier is the plasmid that biological sample comprised of preservation registration number CGMCCNO.0437.It is that the people IL-1 β gene (hereinafter to be referred as IL-1M) that inserts through transforming between the former multiple clone site EcoR I of expression vector pBV220 and BamH I is built into." CCGG " 4 base pairs (bp), pBVIL1 has comprised the pBV220 sequence of all the other 3662bp between pBV220 carrier multiple clone site EcoR I and BamH I, and it has also comprised the insertion sequence IL-1M of 456bp, and total length is 4118bp.The plasmid map of pBVIL1 sees Fig. 1 for details, with first " A " among the EcoR I point of contact GAATCC is the 1st, by the IL-1M (6-461 position) that is followed successively by insertion along the pin direction, the multiple clone site BamH I of pBV220, Sal I, Pst I, Himd III, rrnB rrna transcription termination signal, ammonia benzyl resistant gene (AmpR), c I ts857, and P _RP _LPromotor etc.

The sequence of the IL-1M of described insertion sees Fig. 2 for details, IL-1M is the people IL-1 β gene that 456bp has inserted Xho I and Xba I restriction enzyme site, the 270-275 position of pBVIL1 carrier is an Xho I restriction enzyme site " CTCGAG ", and the 279-284 position is an Xba I restriction enzyme site " TCTAGA ".

PBVIL1 carrier of the present invention has c I ts857 aporepressor gene and the P of pBV220 equally _RP _LPromotor, thereby have the characteristic that thermal induction is expressed, promptly in the time of 42 ℃, the external source segment of inserting pBVIL1 is with IL-1 β gene amalgamation and expression.

According to a second aspect of the invention, pBVIL1 makes up by following technical solution:

1. synthetic primer

Four primers of synthetic of the present invention are respectively F1, R1, F2 and R2 shown in Fig. 3 a.Wherein initiator codon ATG has been introduced at 5 ' end of primers F 1 in F1 and the R1 1-261 position of ripe IL-1 β encoding gene that is used to increase, and has introduced Xho I and Xba I enzyme point of contact at primer R1.F2 and R2 be used to the to increase 285-459 position of ripe IL-1 β encoding gene, primers F 2 has been introduced Xba I site, the introducing of R2 stop code TAA.

2.RT-PCR obtain people IL-1 β gene

From normal people's peripheral blood lymphocytes, extract total mRNA, with above-mentioned F1 and R2 is the upstream and downstream primer, obtained people IL-1 β gene by reverse transcription PCR method (RT-PCR), this process sees people's " IL-1 β and mutant clone thereof, expression and activity research " such as Guo Fukun, Ling Shigan for details, institute of Military Medical Science Institute periodical, 1999,23 (3): 238-9.

3.PCR amplification obtains the IL-1 β gene (IL-1M) of transformation

With people IL-1 β gene is template, be that the upstream and downstream primer carries out pcr amplification with F1, R1 and F2, R2 respectively, the amplification segment is cut with Xba I enzyme respectively, and linked enzyme connects the IL-1M gene that promptly obtains transformation, has promptly introduced the IL-1 β genes of two clones of Xho I and Xba I restriction enzyme site.

As shown in Figure 4, the present invention is on the former working foundation, to be that template increases with plasmid pBV220/IL-1 β.As known to the those skilled in the art, can also can be by the RT-PCR synthetic from the human cDNA library as the people IL-1 β gene of template, also can be the plasmid DNA that contains people IL-1 β gene.

4. gene clone is gone into the pBV220 carrier

The IL-1M gene is cut with EcoR I and BamH I enzyme, inserted the pBV220 carrier of cutting with same enzyme, can obtain to contain the expression vector pBVIL1 of required clone's restriction enzyme site.

According to a third aspect of the present invention, pBVIL1 can be advantageously used in the useful component in gene clone, antigen presentation and the preparation antigen composition.

As shown in Figure 6, pBVIL1 can be used for the insertion and the expression of single goal gene.As long as goal gene contains the restriction enzyme site identical with carrier, just can with inserting the pBVIL1 carrier of handling equally behind the goal gene double digestion, behind the transformed into escherichia coli, can obtain the amalgamation and expression of goal gene and IL-1M by engineered ordinary method.Goal gene can be the minigene fragment of chemosynthesis, also can be the product of pcr amplification.

PBVIL1 can also be used for the common insertion and the expression of various objectives gene.By designing special general connection primer L1, L2 (Fig. 3 b), can realize inserting the purpose of a plurality of genes.As shown in Figure 7, pBVIL1/a and pBVIL1/b are respectively the plasmid that contains goal gene a and b.After cutting pBVIL1/a with Xba I and BamH I enzyme, have on the plasmid that cuts and contain the sticking terminal a gene of Xba I, with pBVIL1/b is template, with general connection primer L1 and R2 amplification, then introduce restriction enzyme site Spe I at the 5 ' end of gene b, because the end of a gene contains and Spe I complementary Xba I sequence, a is with after b is connected, the restriction enzyme site that is used to connect disappears, and the sequence of a and b does not change, and the new clone's restriction enzyme site that contains a and the fusion expression plasmid of b sequence is identical with former expression plasmid.Equally, also can select for use universal primer F1 and primer L2 amplification to have a gene in Sal I site, cut pBVIL1/b with EcoR I and Xho I enzyme, after generation had the b gene of Xho I, the two connected the novel plasmid that also can obtain having a and b gene.According to this thinking, gene is inserted in can also use the same method design primer and continuation, realizes a plurality of heterogeneic amalgamation and expressions with this.Being inserted into gene can be plasmid DNA, also can be gene fragment.

Characteristics of the present invention and advantage are as follows: at first, people IL-1 β is a human body self protein, the antibody of not anti-people IL-1 β in human serum, and the small-molecular peptides that obtains by the pBVIL1 carrier and the non-specific responding of IL-1M fusion expressed product are very low.Secondly, IL-1 β is the good immuno-stimulating factor, but certain toxicity is arranged, the present invention is by transforming G ring relevant with receptors bind in the IL-1 beta molecule and inserting foreign gene, reduced the activity of people IL-1 β, but bioactive peptide sheet in the molecule (ripe molecule 47 to 55) still keeps the immunity of appropriateness to irritate activity, can be used as the immunocompetence that immunological adjuvant improves vaccine in fusion rotein.Moreover the cloning site of pBVIL1 carrier has complementary restriction endonuclease, can carry out intergenic interconnecting easily, is the ideal carrier of antigen presentation therefore.

Below in conjunction with accompanying drawing the specific embodiment of the present invention is described in further detail.

Fig. 1 is structure and the restriction enzyme site figure of expression vector pBVIL1;

Fig. 2 is for inserting the IL-1M gene order of pBVIL1 carrier

Fig. 3 be vector construction, gene synthetic be connected in need the synthetic primer;

A. the general connection primer of the primer b. c. gene synthetic primer of vector construction

Underscore partly is the restriction enzyme site of introducing among the figure

Fig. 4 makes up synoptic diagram for the pBVIL1 fusion expression vector;

Fig. 5 cuts evaluation and changes the evaluation of expressing behind the structure for the enzyme of carrier pBVIL1;

The a/ enzyme is cut evaluation (EcoR I, the BamH I is two cuts)

(1) molecular weight standard: 1857; 1060,929,383,121

(2) PBV220 contrast (3), (4) PBVIL1 plasmid

The SDS-PAGE of the full bacterium of abduction delivering of b/ carrier identifies

(1) IL-1 β expresses band in molecular weight standard (2) carrier

Fig. 6 is the insertion carrier pBVIL1 method synoptic diagram of goal gene;

Fig. 7 goes up interconnection synoptic diagram between the various objectives gene for carrier pBVIL1;

Fig. 8 is the evaluation that different genes expression plasmid PCR and product are expressed;

The PCR of a/ different genes expression plasmid identifies (the primer R of F1 and each gene)

(1)pBVIL1/NS4(2)pBVIL1/C(3)pBVIL1/NS3

(4)pBVIL1/NS5a(5)pBVIL1/NS5b(6)pBVIL1/NS5a-NS5b

(7)pBVIL1/NS4-C(8)pBVIL1/NS4-C-NS5a

(9) pBVIL1-NS4-C-NS5a-NS5b (10) molecular weight standard

The SDS-PAGE of b/ different genes expression plasmid abduction delivering whole cell identifies

(1) pBVIL1 carrier (2) pBVIL1/NS4 (3) pBVIL1/C

(3)pBVIL1/NS3(5)pBVIL1/NS?5a(6)pBVIL1/NS5b

(7)pBVIL1/NS5a-NS5b????(8)pBVIL1/NS4-C

(9)pBVIL1/NS4-C-NS5a???(10)pBVIL1-NS4-C-NS5a-NS5b

(11) molecular weight standard

Fig. 9 is IL-1 beta molecule structure and restriction enzyme site insert division synoptic diagram;

Embodiment:

For further specifying pBVIL1 expression vector, structure and application, describe with reference to the following example, these embodiment do not limit the present invention in any way for explanation.

One, inserts the selection of determining to reach restriction endonuclease at position

By manned IL-1 molecular crystal X-ray diffraction conformational analysis data (network address: www.ncbi.nlm nih.gov/entrez/3d) under the internet, and dynamic observe the molecular structure of people IL-1 β with RasMol, can see that the IL-1 beta molecule is mainly the β barrel-like structure that a plurality of antiparallel β lamellas form, be positioned at the surface of molecule at molecule 86-100 place for the amorphous bent district of circle (G ring), can be used for the toxicity (as Fig. 9) that IL-1 β was inserted and reduced to fragment.

The restriction enzyme site of introducing among the people IL-1 β need satisfy following requirement: do not exist on people IL-1 β inside and pBV220 carrier; Sticking end after enzyme is cut and other restriction endonuclease have complementarity, so as the gene of enzyme after cutting with no longer cut after the gene of cutting with complementary enzyme is connected, realize the segmental connection of a plurality of different genes thus.By the restriction enzyme site analysis, selected Xba I and Xho I as the clone's restriction enzyme site that inserts IL-1 β, they respectively with Spe I and the complementation of Sal I.

Two, the structure of pBVIL1

1. design of primers

According to amplification needs and the restriction enzyme site that will introduce, the primer of having mentioned four fronts has been synthesized in design, and (figure a).F1 and the R1 ripe IL-1 β of the people gene 1-261 position that is used to increase wherein, F2, the R2 IL-1 β gene 285-459 position that is used to increase.

2.RT-PCR obtain people IL-1 β gene:

Separating monocytic cell from normal people's peripheral blood is with 10 ⁶Individual/ml cell concn was cultivated 4 hours at the RPMI-1640 moderate stimulation of 10% calf serum and an amount of LPS, cleer and peaceful suspension cell is abandoned in suction, add denaturing soln (4mol/L guanidine thiocyanate, 25mmol/L Trisodium Citrate pH7.0,0.5%Sarcosyl, 0.1mol/L 0.75ml mercaptoethanol) makes lysis; The sucking-off lysate, and then press " molecular cloning " (Science Press, second edition) method and carry out the total mRNA of extracting and purifying cell.

Get the total mRNA of purifying cells and carry out the synthetic article one chain of reverse transcription, reaction system is: 5 * damping fluid, 8 μ l, the total mRNA10 μ of cell g, dNTPs (10mM) 4 μ l, Rnsin (500U/ μ l) 1 μ l, primer R24 μ l, AMV reversed transcriptive enzyme (8U/ μ l) 3 μ l, add water to 40 μ l, 42 ℃ were reacted 60 minutes.And then pcr amplification, amplification system is: above-mentioned reverse transcription reaction liquid 20 μ l, and primers F 12 μ l, 10 * PCR damping fluid, 10 μ l, Taq archaeal dna polymerase 1 μ l adds water to 100 μ l.Pcr amplification reaction: 94 ℃ 1 minute, 55 ℃ 1 minute, 72 ℃ 1 minute, totally 30 the circulation.With a small amount of PCR product purification test kit (magnificent Shun bio-engineering corporation produce) by specification method purifying.

Plasmid pBV220 preserves for this chamber, according to the operation of " molecular cloning ", the pBV220 carrier is gone in IL-1 β gene clone, construction recombination plasmid pBV220/IL-1 β.

3. in people IL-1 β gene, introduce Xho I and two restriction enzyme sites of Xba I

With plasmid pBV220/IL-1 β is template, carries out pcr amplification with F1, R1 and F2, R2 primer respectively.Amplification system is: contain 10 * amplification buffer, the 10 μ l of magnesium ion, 4 * dNTPs (10mmol), 1 μ l, upstream and downstream primer (Fl, R1 or F2, R2) each 1 μ l, above-mentioned pBV220/IL-1 β 1 μ l, Taq archaeal dna polymerase (3 units/μ l) 1 μ l adds aqua sterilisa to 100 μ l.

Pcr amplification reaction: after 94 ℃ of sex change in 3 minutes, with 94 ℃ 1 minute, 55 ℃ 1 minute, 72 ℃ 1 minute, totally 30 the circulation.With a small amount of PCR product purification test kit (magnificent Shun bio-engineering corporation product) by specification method purifying.

Two kinds of purified products are all cut with Xba I enzyme.The enzyme system of cutting is: 10 μ l10 * enzyme cutting buffering liquids (H), and water 21 μ l, purified product 30 μ l, enzyme Xba I 3 μ l (10 units/μ 1) add water to 60 μ l, 37 ℃ of water-baths 8 hours.Enzyme is cut after product and is separated with agarose gel electrophoresis respectively, reclaims test kit (magnificent Shun bio-engineering corporation product) by specification method recovery with centrifugal a small amount of glue.

Two fragments connect: linked system is: purifying reclaims each 2 μ l of product, and 10 * T4 DNA connects damping fluid 1 μ l, the T4 dna ligase 1 μ l of 12 μ/μ l, and water 6 μ l, 16 ℃ of water-baths are spent the night.The allosteric IL-1 β (IL-1M) that has inserted restriction enzyme site with the connection product is a template, carries out the pcr amplification second time with primers F 1 and R2, and the purifying of product is the same.

4.IL-1M insert pBV220

Double digestion: the IL-1M of above-mentioned purifying and the plasmid pBV220 that extracts with alkaline denaturation, use EcoR I and BamH I double digestion respectively.The PCR product of 30 μ l purifying and 10 μ l plasmids are carried out enzyme respectively cut, the enzyme system of cutting also has 10 * damping fluid (H), 4 μ l, the EcoR I 2 μ l of 10u/ μ l, and the BamH I 2 μ l of 10u/ μ l add water to cumulative volume 40 μ l, 37 ℃ of water-baths 8 hours.Enzyme is cut back difference purifying as stated above.

Insert and connect: linked system is: plasmid pBV220 and IL-1M behind the double digestion, each 1 μ l, 10 * T4 DNA connect damping fluid l μ l, and T4 dna ligase (12u/ μ l) 1 μ l adds water to cumulative volume 10 μ l, and 16 ℃ of water-baths are spent the night.

5. transform and express:

The HB101 colibacillus preserves for this chamber, behind the recovery activation refrigerated bacterial strain, by the CaCl of " molecular cloning " (Science Press, second edition) ₂Method prepares competent cell; Get competent cell 100 μ l, add and connect product 5 μ l, rotate gently with the mixing content, in ice bath, placed 30 minutes, transfer to immediately in 42 ℃ of water-baths and placed 2 minutes, every then pipe adds the not LB substratum of added with antibiotic of 0.5ml, and 37 ℃ of water-baths are after 15 minutes, 37 ℃ of shaking table jogs 45 minutes; Get 100 μ l and transform thalline, evenly coat on the Agar Plating that contains 100mg/ml ammonia benzyl, after room temperature is dried, be inverted overnight incubation for 37 ℃.

6. the evaluation of carrier

Choose the colony of growth from plate, the extraction plasmid enzyme restriction is identified, cut with EcoR I and BamH I enzyme, the 5a that the results are shown in Figure with the evaluation of 1.5% agarose gel electrophoresis, therefrom can see, the control plasmid of the 2nd swimming lane shows as the plasmid band of 40kb size, because the small segment between two restriction enzyme sites is too little, therefore on electrophoretogram, can't see 3,4 swimming lanes are after IL-1M inserts, the enzyme of plasmid pBVIL1 is cut the result, except that the carrier of 40kb, also can see the IL-1M band of about 460bp, proof IL-1M has inserted pBV220, and promptly pBVIL1 successfully constructs.Fig. 5 b is for transforming the bacterium that the pBVIL1 carrier is arranged, change 42 ℃ of inducing culture 2.5 hours 32 ℃ of cultivations over to after 2 hours, get whole cell and make polyacrylamide gel electrophoresis (SDS-PAGE), resolving gel concentration is 15%, " molecular cloning " seen in operating process, the result has an engrain band at the 17kd place as shown in the figure, shows that inserting IL-1M has obtained expressing efficiently.

Three, the purposes of pBVIL1

(1). the insertion of goal gene and and IL-1M amalgamation and expression

1. insert

Design the different piece of different primer amplification HCV encoding genes in the experiment, the primer sequence of design is seen Fig. 4 c, C-F wherein, C-R is used to increase, and (the HCV sequence with GenBank sequence " HPCCGAA " by name is as the criterion 68 of 22-1, HCV/C antigen encoding gene down together), NS3-F, NS3-R be used to the to increase HCV/NS3 helicase encoding gene of 3574-4371 position, NS4-F1, NS4-R1, NS4-F2, NS4-R2 is used for the terminal encoding gene of HCV/NS4C of synthetic 5746-5841 position, NS5a-F, NS5a-R be used to the to increase HCV/NS5a encoding gene of 6796-6942 position, NS5b-F, NS5b-R be used to the to increase encoding gene of 7141-7374 position.

With the example that is inserted as of HCV/NS4 gene because the 3 ' end of primer NS4-F1 and NS4-R1 has complementary sequence, under appropriate condition behind the complementary sequence hybridization, in the PCR reaction, the two each other template increase.The amplification system of experiment and PCR reaction are provided with embodiment two described, and cycle number is 5.Get the 1 μ l amplified production conduct template of PCR once more, add primer NS4-F2 and NS4-R2, carry out 30 round-robin pcr amplifications again.Pcr amplification product is purified, with Xho I and Xba I double digestion, plasmid pBVIL1 is also used identical enzyme double digestion, gene after enzyme is cut is connected with plasmid is the pBVIL1/NS4 expression plasmid, and transform then, expression etc. operated all described with embodiment two.

Different genes source and size that table 1. is expressed with pBVIL1

The polypeptide title	The gene template source	Clip size
			???HCV/NS4 ???HCV/C ???HCV/NS3 ???HCV/NS5a ???HCV/NS5b	Chemosynthesis plasmid pBRTM/HCV plasmid pEX/NS3 plasmid pBRTM/HCV plasmid pBRTM/HCV	????32?aa ????49?aa ???266?aa ????49?aa ????78?aa

According to above-mentioned steps, see Table 1 with the gene segment of different primer amplifications, insert and be built into different expression plasmids in the pBVIL1 carrier and be respectively pBVIL1/NS4, pBVIL1/C, pBVIL1/NS3, pBVIL1/NS5a, pBVIL1/NS5b.

2. the PCR of expression plasmid identifies

With the HCV/NS4 gene is example, with plasmid pBVIL1/NS4 behind the purifying is template, with primers F 1 is forward primer, NS4-R2 carries out pcr amplification for the negative sense primer, amplification condition embodiment two, 1.5% agarose gel electrophoresis of amplified production the results are shown in Figure 8a1, can amplify the special band of about 370bp, proves that the NS4 gene inserts.Other authentication method that respectively inserts gene plasmid is identical, and only used negative sense primer is respectively primer C-R, NS3-R, NS5a-R, NS5b-R (Fig. 3 c), electrophoresis is identified and is seen Fig. 8 a band 2-5, amplify corresponding fragment and be followed successively by 420bp, 1000bp, 420bp and 510bp.

3. the SDS-PAGE of genetic expression identifies

With the HCV/NS4 gene is example, the qualification process of genetic expression is as follows: get and insert HCV/NS4 expression of gene plasmid pBVIL1/NS4, by embodiment two methods transform, cultivation and thermal induction cultivate, get whole cell and carry out SDS-PAGE (gum concentration 15%) qualification result such as Fig. 8 b: the district is with 2 an engrain band is arranged about the 22kd place, and is consistent with the theoretical molecular after the fusion of NS4 and IL-1M.Other each gene HCV/C, NS3, NS5a, the authentication method that NS5b expresses is identical, and district band 3-6 is each plasmid expression bacterium among Fig. 3 b, and the molecular weight of expressing band is respectively: 25kd, 50kd, 25kd and 29kd, the result is consistent with the expection molecular weight.

(2). the connection of various objectives gene and amalgamation and expression

Interconnecting between different genes is by designing special general connection primer L1, L2 (Fig. 3 b), can carry out in containing between various objectives expression of gene plasmid of having built.We have made up 4 kinds of expression plasmids that contain the polygene connection as shown in table 2.

Now with the example that is configured to of pBVIL1/NS4-C expression plasmid:

Get the pBVIL1/NS4 plasmid 10 μ l of purifying, carry out double digestion with Xba I and BamH I by embodiment two described methods; Meanwhile, getting plasmid purification pBVIL1/C is template, and (Fig. 3 a) carries out pcr amplification (method is with embodiment two) with general connection primer L1 (Fig. 3 b) and primer R2.Make then that gene after enzyme is cut is connected with plasmid, operations such as conversion and abduction delivering are all described with embodiment two.

The expression plasmid that contains the polygene connection that table 2. makes up with carrier pBVIL1

Make up the plasmid title	Be used for digested plasmid	Be used for template plasmid
			PBVIL1/NS4-C PBVIL1/NS5a-NS5b PBVIL1/NS4-C-NS5a PBVIL1/NS4-C-NS5a-NS5b	PBVIL1/NS4 PBVIL1/NS5a PBVIL1/NS4-C PBVIL1/NS4-C-NS5a	PBVIL1/C PBVIL1/NS5a PBVIL1/NS5a PBVIL1/NS5b

Other NS5a-NS5b, NS4-C-NS5a, the cloning process of NS4-C-NS5a-NS5b gene is identical, and different is plasmid that used enzyme cuts is different with the plasmid that is used to do pcr template, and table 2 is each fused antigen expression plasmid used respectively plasmid situation when making up.

The PCR authentication method of plasmid is as described in the embodiment three (), be template with plasmid behind the purifying respectively, with primers F 1 is forward primer, the negative sense primer (four kinds of polygene of table 2 connect primer C-R, NS5b-R, NS5a-R, the NS5b-R that plasmid is used Fig. 3 c respectively) that connects last gene of gene is that the negative sense primer carries out pcr amplification with each, result such as Fig. 8 a, each plasmid district band 6-9 is respectively NS5a-NS5b, NS4-C, NS4-C-NS5a, NS4-C-NS5a-NS5b all can amplify respective segments, about respectively 660bp, 520bp, 670bp and 910bp.

The evaluation such as embodiment three (one) methods that merge multi-gene expression are carried out, getting whole cell behind the inducing culture carries out the SDS-PAGE qualification result and sees Fig. 8 b, district band 7-10 is respectively NS5a-NS5b, NS4-C, NS4-C-NS5a, the amalgamation and expression situation of many goal gene such as NS4-C-NS5a-NS5b, as seen from the figure at 43kd, 30kd, respectively there is an engrain band at 38kd and 67kd place, and molecular weight is consistent with expected results.

Claims (13)

1. expression vector pBVIL1, wherein said carrier has comprised the most of sequence of plasmid pBV220 and the people IL-1 β gene of transformation.

2. expression vector according to claim 1, wherein said carrier are the plasmid that biological sample comprised of preservation registration number CGMCC NO.0437.

3. expression vector according to claim 1, wherein said carrier are the closed loop plasmid of 4118bp, and first " A " of going up among the EcoR I restriction enzyme site GAATCC with pBVIL1 is the 1st, the people IL-1 β gene of 6-461 position for transforming on the carrier.

4. carrier according to claim 2, most of sequence of wherein said pBV220 are meant 3662 Nucleotide " CCGG " 4 Nucleotide between pBV220 multiple clone site EcoR I and BamH I.

5. carrier according to claim 2, the people IL-1 β gene of wherein said transformation is shown in SEQ ID NO:1, and it contains initiator codon ATG, terminator codon TAA and restriction enzyme site Xho I and Xba I.

6. carrier according to claim 4, the 270-275 position of wherein said carrier are Xho I restriction enzyme site, and the 279-284 position is an Xba I restriction enzyme site.

7. according to the described preparation of expression vectors method of claim 1, it may further comprise the steps:

(1) synthetic primer;

(2) reverse transcription PCR obtains people IL-1 β gene;

(3) pcr amplification obtains the IL-1M gene;

(4) the pBV220 carrier is gone in the IL-1M gene clone.

8. want 7 described preparation methods according to right, the sequence that wherein is used to the primer that increases from 5 ' end is:

R1：

5’GC?TCT?AGA?GGC?CTC?GAG?GGG?ATC?TAC?ACT?CTC?3’

F2:5’GC?TCT?AGA?GAA?AAG?CGA?TTT?GTC?3’

9. according to right 8 described preparation methods, the primer R1 that wherein is used to increase contains Xho I and Xba I restriction enzyme site, and F2 contains Xba I restriction enzyme site.

10. the purposes of expression vector according to claim 1 in gene clone, antigen presentation and vaccine production.

11. purposes according to claim 8, wherein pBVIL1 is used for the insertion and the expression of individual gene.

12. purposes according to claim 8, wherein pBVIL1 is used for the insertion and the expression of a plurality of genes.

13. purposes according to claim 8, wherein pBVIL1 is used to prepare immune vaccine.

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