rhGH-Fc工程细胞的构建及筛选  

Construction and screening of rhGH-Fc engineering cells

作　　者：俞露[1] 刘玉林[1] 申兆兴[1] 刘涵[1] 龚士卿[1] 王莹[1] 吕效宇 王诗琪[1] 张红[1] 刘景会[1] 

机构地区：[1]长春生物制品研究所有限责任公司细胞因子室,吉林长春130012

出　　处：《中国生物制品学杂志》2016年第10期1021-1026,共6页Chinese Journal of Biologicals

基　　金：吉林省科技厅重点科技攻关项目(20160204034YY)

摘　　要：目的研制具有较长半衰期的长效重组人生长激素(recombinant human growth hormone,rhGH),构建并筛选出高表达rhGH-Fc免疫融合蛋白的细胞株。方法将hGH基因与特异位点突变后的人抗体IgG4Fc段用柔性linker连接,克隆至GS双表达载体上,构建重组表达质粒,经两次双酶切鉴定及测序分析后,将构建正确的质粒稳定转染CHO-k1细胞。通过逐渐提高蛋氨酸磺酰胺(methionine sulphoximine,MSX)浓度,ELISA法筛选高表达的细胞株;利用亲和层析初步纯化目的蛋白,并进行SDS-PAGE、HPLC、等电聚焦电泳分析;通过大鼠去垂体法测定长效rhGH的生物学活性,比较本课题研制的长效GH与其他普通GH的半衰期。结果重组质粒phGH-Fc-1经两次双酶切及测序鉴定,证明构建正确;成功筛选出高表达rhGH-Fc融合蛋白的细胞株,表达量可达1g/L;纯化的目的蛋白相对分子质量大小及等电点均与理论值一致,纯度可达95%以上;经大鼠去垂体体内活性测定,其比活为1.1IU/mg,依大鼠体重增长情况判断,其生物半衰期高于普通rhGH。结论成功构建并筛选出高表达长效rhGH的工程株,为后续长效rhGH的研制奠定了基础。Objective To prepare a recombinant human growth hormone（rhGH） with long half-life, and construct and screen a cell line for high expression of long-acting rhGH. Methods The h GH gene was connected to the human IgG Fc fragment with mutations in specific sites by a flexible linker, and cloned into GS double expression vector. The constructed recombinant plasmid was identified by restriction analysis and sequencing, and stably transfected to CHO-k1 cells. The cell strains for high expression were screened by MSX pressure screening and ELISA. The target protein was preliminarily purified by affinity chromatography, and analyzed by SDS-PAGE, HPLC and isoelectric focusing electrophoresis. The biological activity of long-acting recombinant h GH was determined by in vivo activity test in rats, while half-life was compared with those of other common GH. Results Restriction analysis and sequencing proved that recombinant plasmid phGH-Fc-1 was constructed correctly. The cell strain for high expression of rhGH-Fc fusion protein was successfully screened, in which the expression level of target protein reached 1g/L. Both the relative molecular mass and isoelectric point of purified target protein were consistent with those in theory, while the purity reached more than 95%. In vivo activity test in rats showed that the specific activity of target protein was 1. 1 IU / mg. According to the weight gain of rats,the biological half-life of rhGH was longer than those of common rhGH. Conclusion The engineering cell line for high expression of long-acting rhGH was constructed and screened successfully, which laid a foundation of further study on long-acting rhGH.

关 键 词：人生长激素 Fc片段 免疫融合蛋白 GS筛选系统 半衰期 长效 

分 类 号：Q786[生物学—分子生物学]