IL-12 expression carrier as well as eukaryotic cell strain expressed thereby and uses thereof

A technology of expression vectors and eukaryotic cells, applied in the direction of cells modified by introducing foreign genetic material, applications, medical preparations containing active ingredients, etc., can solve problems such as difficult screening

Active Publication Date: 2008-06-18
广州市茵良强生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Aiming at the problem that the expression of the two subunits in the existing rhIL-12 does not match, and it is usually difficult to screen for high-expression target clones after transfe

Method used

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  • IL-12 expression carrier as well as eukaryotic cell strain expressed thereby and uses thereof
  • IL-12 expression carrier as well as eukaryotic cell strain expressed thereby and uses thereof
  • IL-12 expression carrier as well as eukaryotic cell strain expressed thereby and uses thereof

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Experimental program
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Effect test

Embodiment 1

[0032] Primer design and gene amplification

[0033] Design the following p35 subunit amplification primers and p40 subunit amplification primers (entrusted to Shanghai Sangon Bioengineering Co., Ltd. to synthesize), use the RNeasy extraction kit (Qiagen company product) to extract the total RNA of human cells, and use it as a reverse transcription PCR Templates were subjected to one-step RT-PCR amplification (Takara kit), to obtain a p35 gene product with a length of 684bp and a p40 gene product with a length of 1002bp (see figure 1 ), which are consistent with the expected size.

[0034] The sequence of the P35 forward primer is (the underline indicates the SalI restriction site):

[0035] 5'-TGG GTC GAC GCCACCATGTGTCCAGCGCGCAGCCTC-3'

[0036] The sequence of the P35 reverse primer is (the underline indicates the SalI restriction site):

[0037] 5'-TGG GTC GAC TCAGGAAGCATTCAGATAGC-3'

[0038] The sequence of the forward primer of P40 is (the underline indicates the r...

Embodiment 2

[0043] gene cloning

[0044] (1) Construction of recombinant plasmid pDouble-P35

[0045] Vector pDouble carries the double expression cassette. The PCR product of the p35 gene was cloned and inserted into the pDouble vector after single-digestion with SalI, and the recombinant plasmid inserted in the forward direction was screened and named pDouble-P35 (see figure 2) .

[0046] (2) Construction of recombinant plasmid pP40IRESneo

[0047] The vector pIRESneo carrying the Neo resistance selection marker was purchased from Clontech, USA. The PCR product of the p40 gene was digested with BamHI and cloned into the pIRESneo vector, and the recombinant plasmid inserted in the forward direction was screened and named pP40IRESneo (see image 3 ).

[0048] (3) Construction of recombinant plasmid p75

[0049] The vector pP40IRESneo was double-digested with NruI+XhoI, and the expression cassette carrying the entire P40-IRES-Neo was transferred into the pDouble-P35 vector after sin...

Embodiment 3

[0051] Gene identification and sequencing

[0052] Identification of recombinant plasmids pDouble-P35, pP40IRESneo and pP75

[0053] (1) pDouble-P35 was digested and identified with EcoRI and BglII single enzyme digestion, and two bands (EcoRI single enzyme digestion) with a size of about 2.3kb and 4.4kb were obtained, and two bands with a size of about 1.3kb and 5.4kb were obtained. Band (BglII digestion), the result was consistent with the expectation ( Figure 5 ).

[0054] (2) pP40IRESneo was digested and identified with KpnI and EcoRI, respectively, and two bands with sizes of about 1.9 kb and 4.3 kb (KpnI single enzyme digestion) and two bands with sizes of about 0.8 kb and 5.4 kb were obtained (EcoRI single enzyme digestion), the results were consistent with expectations ( Figure 6 ).

[0055] (3) pP75 was digested and identified by EcoRI and EcoRV single enzyme digestion, and four bands (EcoRI digestion) with sizes of about 0.76kb, 1.04kb, 2.25kb and 5.37kb were o...

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Abstract

The present invention relates to a novel method of using mammalian cell efficient expression to recombine human interleukin 12 (rhIL-12). The present invention transcripts two p35 subunits and one p40 subunit of rhIL-12 to be constructed inside a same carrier; each subunit is processed for expression level regulation by a transcription regulating element, and the efficient expression of rhIL-12 is realized by introducing the regulating elements of Intron and SV40Enhancer which can improve the expression level.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular the invention relates to the construction of an interleukin-12 high-efficiency eukaryotic expression vector, the transfection of mammalian cells, the screening and identification of rhIL-12 high-efficiency expression cell lines and the preparation of rhIL-12 and Pharmaceutical applications. Background technique [0002] rhIL-12 (recombinant human interleukin 12, rhIL-12) is a glycoprotein with a molecular weight of 75KD, which is a heterodimer formed by two subunits P35 and P40 linked by disulfide bonds. IL-12 has anti-virus, anti-tumor and tumor metastasis capabilities, mainly because it can activate cytotoxic T lymphocytes ((Brunda et al. (1993) J. Exp. Med. 178, 1223-1230; ), and stimulate The production of IFN-γ is related to (Nastala, C.Journal of Immunology, 1994; 153:1697-1706; Seder, R., Proc.Natl.Acad.Sci.USA, 1993; 90:10188-10192). Currently known IL -12 is a good initiating...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/24C12N15/85C12N5/10A61K38/20
Inventor 粟宽源柴玉波刘勇李鼎峰夏书奇曾振飞
Owner 广州市茵良强生物科技有限公司
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