CN1273601C - Expression carrier for high-efficient screening target protein, its preparation method and use - Google Patents

Abstract

The present invention discloses an expression carrier for the high-efficiency sieving of target protein, a preparing method thereof and an application thereof. The inventor leads an internal ribosome entry site in a eukaryotic expression carrier comprising a CMV promoter and a dhfr amplifying system for connecting an anti-erbB2-scFv-Fc gene with an IL-2 fusion gene and a neomycin resistance gene. A PCR method is used for carrying out point mutation to a Neo gene which is constructed to form an expression carrier pCMV-HFI-mNeo; the carrier is characterized in that the coexpression of the fusion protein gene and the Neo gene are started under the same promoter by leading in IRES, and the activation of a Neo gene expression product is weakened by the point mutation of the Neo gene so as to enhance the sieving efficiency of positive expression cloning; moreover, the work amount of sieving is largely reduce, and the stable and high-efficiency expression cloning of a target gene is obtained.

Description

A kind of expression vector of efficient screening target protein, Preparation Method And The Use

Technical field

The present invention relates to a kind of engineering carrier, relate in particular to a kind of expression vector of efficient screening target protein, also relate to Preparation Method And The Use.

Background technology

The biotechnology of antibody has great research and using value, also has huge market potential simultaneously.Efficiently expressing of antibody is the bottleneck technology of genetic engineering antibody preparation always.Improve antibody expression output, reduce its production cost and become the problem that presses for solution at present.Efficiently expressing of gene depends on many factors in mammalian cell, comprises transcribing, translate controlling elements, RNA metabolism, gene copy number, mRNA stability, the location of gene on host cell chromosome etc.Present most of carrier for expression of eukaryon is the random integration carrier, and the sequence of both sides, gene integration site can greatly influence the expression intensity of goal gene, usually have the integration site more than at least 15000 in the mammalian cell karyomit(e), the high-expression clone of desiring to filter out a few optimum integration site is extremely difficult.Adopt the method for regulating the selected marker gene expression intensity to screen, reduce the expression level of selected marker gene, help screening the high expressing cell clone expressing vector integration site.When adopting neomycin resistance gene (Neo gene) as positive selected marker gene, the a small amount of expression just can make cell pass through screening, and it is carried out point mutation, this mutator gene only is incorporated into the host cell gene group chromosome and expresses active region (focus), its expression product amount could be resisted the screening of G418, transfectional cell could be survived, thereby helps the cell clone that acquisition fast efficiently expresses goal gene.When Yenofsky etc. sport D to the 182E of the neomycin phosphotransferase II (NPTII) of Neo genes encoding in prokaryotic expression carrier, discovery contains under the various G418 concentration that the E.coli cell of normal Neo gene adopts in test all well-growns, and the cell growth conditions when G418 concentration is 20 μ g/ml that contains sudden change Neo gene is not good, when being 30 μ g/ml, concentration do not have growth (Yenofsky, R., Fine, M., and Pellow, J.W. (1990) A mutant neomycin phosphotransferase gene reduces the resistance oftransformants to antibiotic selection pressure.Proc.Natl.Acad.Sci.USA87:3435-3439); Blazquez J etc. has made up multiple prokaryotic expression carrier mutant, comprise 36V → M, 188H → Y, 88H → S, 188H → L, 190G → D, 212R → G, 262D → N, found that with wild-type Neo genophore and compare, kantlex is respectively 6.2% of wild-type to the minimum inhibitory concentration of various mutant, ≤ 0.2%, 0.4%, 3.1%, ≤ 0.2%, 1.5% and 1.5% (Blazquez, J., Davies, J., Moreno, F. (1991) Mutations in the aphA-2gene of transposon Tn5mapping within the regions highly conserved in aminoglycoside-phosphotransferases strongly reduce aminoglycoside resistance.Mol.Microbiol.5 (6): 1511-1518.); Gao Chuan etc. sport M with 36V and find with the Neo gene mutation body to serve as that the screening rate of carrier for expression of eukaryon of screening marker gene is apparently higher than control group, the high expression level activity of the cell colony luciferase that is screened is 25 times of (Gao Chuan of normal Neo gene transfecting cell colony, Zhu Xudong, Zhou Xiaowei, in virtue, Lu Baisong, Huang Peitang. with the neomycin resistance gene mutant is the Construction of eukaryotic of screening sign. the biotechnology journal.2002，18(3)：308-312)。In carrier for expression of eukaryon, do not see at present the report of using at Neo gene 546 site mutations is arranged.

Summary of the invention

For screening efficiency and the expressing protein output that increases the recombination fusion protein stable clone, the invention discloses a kind of expression vector pCMV-HFI-mNeo, this carrier is the two-cistron expression vector that contains Anti-Her2-scFv-Fc and IL-2 gene Fusion gene and neomycin resistance gene mutant mNeo, and this carrier is to be that the basis changes structure and obtains with carrier PCI-neo.

Carrier of the present invention is by introducing the coexpression that internal ribosome entry site IRES (genbankgi|12657334|emb|AJ310442.1|CVE310442) realizes antigen-4 fusion protein gene and mNeo gene, fusion gene wherein is Anti-Her2-scFv-Fc gene and IL-2 gene Fusion gene, the mNeo gene is that the 546th the guanine of neomycin resistance gene Neo sports thymus pyrimidine, and the 182nd glutaminic acid residue of its coded protein sports the aspartic acid residue.

The preparation method of carrier of the present invention is as follows:

At first on the pCI-neo carrier, replace neo gene, carrier construction pCMV-dhfr with dhfr gene (genebank NM_010049); Insert the anti-c-erbB2-scFv gene, IgG1 Fc fragment gene and IL-2 gene are formed Anti-Her2-scFv-Fc and IL-2 gene Fusion gene, carrier construction pCMV-HFI; Introduce IRES sequence and Neo gene then on the pCMV-HFI carrier, fusion gene is connected by the IRES sequence with the Neo gene, carrier construction pCMV-HFI-Neo; Be template by PCR method with Neo gene 526-560bp then, introduce a point mutation at 5 ' end primer and clone this sequence that this sequence and former sequence are replaced, and formation contains Neo gene mutation body carrier pCMV-HFI-mNeo.

The invention also discloses the application of expression vector pCMV-HFI-mNeo in efficient screening target protein.

(1) cell cultures, transfection and screening: the plasmid vector that makes up is cultivated by plasmalogen mediated method transfectional cell, detect fusion protein expression with the ELISA method, it is big and expression amount is high that the clone that the result picks out contains sudden change Neo gene contains clone's ratio of expressed fusion protein among the clone of normal Neo gene, the different G418 concentration gradients that are cloned in of stably express add the not variation of level of depressing its expressing protein, and the growth conditions of cell is good.

(2) transfectional cell is to the resistance of G418: transfectional cell is cultivated with the G418 pressurization, under the identical G418 concentration, obviously is less than with the resistance clone of pCMV-HFI-mNeo transfectional cell and uses the pCMV-HFI-Neo cells transfected; The G418 of different concns is to the pressurization of pCMV-HFI-mNeo transfectional cell, and clone's number that G418 concentration obtains when high is far smaller than the clone's number that obtains when concentration is hanged down, and shows that the toxic ability of sudden change Neo gene expression product opposing G418 significantly weakens.

(3) RT-PCR analyzes: the fusion rotein of sudden change Neo gene transfecting cell and Neo gene transcription level are than the cell height that contains normal Neo gene.

With 182 L-glutamic acid rite-directed mutagenesises of neomycin phosphotransferase II (NPT II) of neomycin resistance gene (Neo gene) coding is that aspartic acid causes the active decline of NPT II, and does not influence mRNA and the proteic stability of NPT II.The toxic ability of resistance clone opposing screening of medicaments G418 that the ratio that contains sudden change Neo gene has normal Neo gene weakens, this gene only is incorporated into the host cell gene group chromosome and expresses active region (focus) ability effective expression, transfectional cell could be survived, thereby helps the clone that acquisition fast efficiently expresses goal gene.

The present invention is based on the efficient screening of sudden change Neo gene and expresses the carrier for expression of eukaryon that target protein is cloned, its advantage is that (1) is by introducing ribosome entry site(RES) IRES between antigen-4 fusion protein gene and Neo gene, two genes are transcribed under same promotor, be built into two-cistron expression vector, realize that the coupling of antigen-4 fusion protein gene and Neo gene is expressed; (2) by Neo gene expression product activity that the Neo gene mutations is weakened, thereby improved positive expression clone's screening efficiency, significantly reduced the screening operation amount, obtained the stability and high efficiency cloning by expression of goal gene, realized the purpose of efficient rapid screening to the cell clone of high-expression target proteins.

The shortcoming that wastes time and energy when the present invention has overcome screening and cloning has in the past been saved the time, human and material resources and financial resources greatly.With carrier transfection CHO cell of the present invention, can obtain to stablize the cell clone of high expression level fusion rotein fast, screening efficiency and the antibody expression output that improves high expression level recombinant protein clone is had using value preferably.

Description of drawings

Fig. 1 makes up schema for expression vector pCMV-HFI-mNeo.

Fig. 2 is a pCMV-HFI-mNeo two-cistron expression vector structure iron.Reorganization anti-erbB2-scFv-Fc-IL2 antigen-4 fusion protein gene links to each other by an IRES fragment with sudden change Neo gene and is controlled by the SV40 promotor by CMV promotor startup dhfr gene.

Fig. 3 sports thymus pyrimidine for the guanine of 546 of neomycin resistance genes, and the L-glutamic acid that its proteins encoded is 182 sports aspartic acid.

Fig. 4 is the fusion rotein ELISA detected result that contains the clonal expression of normal Neo gene.

Fig. 5 is the fusion rotein ELISA detected result that contains the clonal expression of sudden change Neo gene.

Fig. 6 contains the fusion rotein ELISA detected result of expressing under the different G418 pressurization concentration of being cloned in of sudden change Neo gene.

Fig. 7 forms analysis chart for the clone.With pCMV-HFI-Neo and pCMV-HFI-mNeo transfection CHO cell, under 300 μ g/ml G418 concentration, pressurize, the clone who forms after 10 days dyes with Rui Shi-Ji's nurse Sa.Wherein A is the pCMV-HFI-Neo transfection; B is the pCMV-HFI-mNeo transfection.

Fig. 8 is that different G418 concentration form clone's the figure that influences to it behind the pCMV-HFI-mNeo transfection CHO cell.Wherein the G148 concentration of A is 200 μ g/ml, and the G148 concentration of B is 400 μ g/ml, and the G148 concentration of C is 600 μ g/ml, and the G148 concentration of D is 800 μ g/ml.The clone dyes with Rui Shi-Ji's nurse Sa

Fig. 9 is the RT-PCR gel electrophoresis analysis figure of genetic transcription.Wherein 1,4 is human IgG Fc gene; 2,5 is the Neo gene; 3,6 is the GAPDH gene; 7 is DL2000DNA marker.The mRNA of Neo for from the pCMV-HFI-Neo cells transfected, extracting, the m RNA of mNeo for from the pCMV-HFI-mNeo cells transfected, extracting.

Embodiment

The structure of embodiment one pCMV-HFI-mNeo carrier

1, material

Coli strain, Chinese hamster ovary celI are the international standard strain, preserve this chamber, the PCR primer is synthetic to be finished by the rich inferior biological company limited in Shanghai with sequencing, T4DNA ligase enzyme, DMEM substratum are GibcoBRL company product, Vent archaeal dna polymerase, nucleic acid restriction endonuclease are Biolab company product, plasmid extraction kit is the vast Imtech in a Beijing product, it is Dalian precious biotechnology company limited product that dna fragmentation reclaims test kit, the goat anti-human igg is this chamber self-control, and the goat anti-human igg of horseradish peroxidase-labeled is available from Beijing Zhong Shan biotech company.Carrier pCI-neo is available from Promega company, and anti-c-erbB-2 antibody is light, heavy chain variable region gene (Genbank:A22466; A22467) provide by the Dr.Lei Yu of pharmaceutical college of U.S. You Ta university, people's IgG antibody 1 constant region gene (Genbank:AF150959) is provided by the Wang Haitao professor of BIO ENGINEERING INST MILITARY, and IL-2 gene (Genbank:NM_000586) angles from activated T lymphocyte by the RT-PCR method and gets.

2, method and result

At first on the pCI-neo carrier, replace neo gene, carrier construction pCMV-dhfr with dhfr gene (genebank NM_010049) by BamH I and Stu I restriction enzyme site; Between Nhe I and EcoRI, insert the anti-c-erbB2-scFv gene, between EcoR I and Xho I, insert IgG1 Fc fragment gene, between Xho I and Mlu I, insert the IL-2 gene, be built into pCMV-HFI (Shi, M., Xie, Z.G, Feng, J.N., Sun, Y.S., Yu, M., Shen, B.F., Guo, N. (2003) A recombinantanti-erbB2, scFv-Fc-IL-2fusion protein retains antigen specificity and cytokinefunction.Biotechnol.Letters.25:815-819.); Secondly between Mlu I and Xba I, introduce the IRES sequence, by PCR method with pcDNA3.1 (+) be template clone the Neo gene and be inserted into carrier Xba I and Not I between, be built into carrier pCMV-HFI-Neo; Be template by PCR method with Neo gene 526-560bp at last, introduce a point mutation at 5 ' end primer and clone this sequence, clonal mutation Neo gene PCR primer sees Table 1, the PCR reaction conditions is: 95 ℃ of pre-sex change 2 minutes, 94 ℃ 20 seconds, 58 ℃ 20 seconds, 72 ℃ 25 circulations in 30 seconds, 72 ℃ were extended 10 minutes.By BssH II and Not I site this sequence and former sequence replacement formation are contained Neo gene mutation body carrier pCMV-HFI-mNeo (see figure 1) then.Like this, the guanine that the Neo gene is 546 sports thymus pyrimidine, and the L-glutamic acid that its proteins encoded is 182 sports aspartic acid (seeing Fig. 2, Fig. 3).

Table 1PCR primer sequence

Primer title (restriction endonuclease)	Sequence
		Neo 5` end primer (BssHII) Neo 3` end primer (Not I)	5`-ttggcgcgcatgcccgatggcgatgatctcgtcgtga-3` 5`-atagtttagcggccgcctcagaagaactcgtca-3`

The effect of embodiment two pCMV-HFI-mNeo carriers in efficient screening target protein

1, cell cultures, transfection and screening

Chinese hamster ovary cell (CHO) is cultivated in containing the DMEM substratum of 10% foetal calf serum.Plasmid vector advances in the Chinese hamster ovary celI by plasmalogen (purchasing the company in Gibco) mediated method transfection, at 37 ℃ and 5%CO ₂The substratum of cultivating in the incubator after 8 hours antibiotic-free, serum-free changes the DMEM substratum that contains 10% foetal calf serum into, is that the substratum of 300 μ g/mlG418 is replaced normal substratum with containing concentration after 48 hours.Pick out single clone with limiting dilution assay afterwards.Gather in the crops supernatant after at the bottom of cell covers with culturing bottle, culture supernatant detects fusion protein expression with the ELISA method.By 96 orifice plates, the bonded fusion rotein detects with the goat anti-human igg of horseradish peroxidase-labeled with goat anti-human igg's bag, and the OPD colour developing is surveyed absorbance under the 492nm wavelength.The result shows in 12 mono-clonals that contain normal Neo gene of picking out to have only 3 clonal expression fusion roteins, high expression level amount is about 150ng/ml, remaining clone all detects less than the protein expression (see figure 4), and 5 equal high expression level fusion roteins of clone that contain sudden change Neo gene of picking out, expression amount is three times of (see figure 5)s of normal Neo gene transfection clone near 500ng/ml.The clone of stably express further adds the level of depressing its expressing protein in different G418 concentration gradients and does not change (see figure 6), and the growth conditions of cell is good.

2, transfectional cell is to the resistance of G418

Transfectional cell changes into 24 holes and 6 orifice plates, with the G418 pressurization, changes a subculture in 3 days, and the clone who formed in the metapore in 10 days dyes with Rui Shi-Ji's nurse Sa dye liquor.Under identical 300 μ g/ml G418 concentration, pressurize, resistance clone with the pCMV-HFI-mNeo transfectional cell obviously is less than with pCMV-HFI-Neo cells transfected (see figure 7), the activity that sudden change Neo gene expression product is described is obviously weakened, and the clone who only is incorporated into hotspot location high expression level NPT II could survive.Under different G418 concentration, the pCMV-HFI-mNeo transfectional cell is pressurizeed, the result shows that the clone's number that obtains is far smaller than the clone who obtains and counts (see figure 8) when 200 μ g/ml when G418 concentration is equal to or greater than 400 μ g/ml, show that the virulence of sudden change Neo gene expression product opposing G418 significantly weakens.

3, RT-PCR analyzes

Extract the mRNA by pCMV-HFI-Neo and pCMV-HFI-mNeo transfection CHO cell respectively, reverse transcription becomes cDNA, is increase respectively human IgG Fc section, Neo (comprising mutant and wild-type), GAPDH gene of template with cDNA.The PCR product is analyzed (see figure 9) with 1% agarose gel electrophoresis.The result shows that the fusion rotein of sudden change Neo gene transfecting cell and Neo gene transcription level are than the cell height that contains normal Neo gene.

Claims (3)

1, a kind of carrier for expression of eukaryon, this carrier are to be that the basis changes the structure acquisition with PCI-neo, and it is characterized in that: 1. the neo gene is replaced into the dhfr gene in this carrier; What 2. be connected into after CMV I.E. promotor is to be recorded in anti-c-erbB2-scFv gene and the human immunoglobulin gene IgGlFc fragment gene that the GENBANK accession number is A22466 and A22467; 3. be connected into the interleukin II gene after the IgGlFc fragment gene, antibody gene and IL-2 gene constitute fusion gene together; 4. the fusion gene back is connected into the sudden change Neo gene that internal ribosome entry site IRES sequence and the 546th guanine sport thymus pyrimidine; 5. this two-cistron expression vector is named as pCMV-HFI-mNeo.

2, the preparation method of the described carrier of claim 1 comprises the steps:

(1) be the dhfr gene by BamH I and Stu I restriction enzyme site with the neo gene substitution on the pCI-neo carrier, be built into carrier pCMV-dhfr,

(2) between Nhe I and EcoR I, insert the anti-c-erbB2-scFv gene, between EcoRI and Xho I, insert IgGl Fc fragment gene, between Xho I and Mlu I, insert the IL-2 gene, be built into pCMV-HFI,

(3) between Mlu I and Not I, insert IRES sequence and Neo gene, then the Neo gene is carried out the 546th guanine and sport thymus pyrimidine, be built into carrier pCMV-HFI-mNeo at last.

3, the application of the described carrier of claim 1 in screening expression target protein clone.

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