基因工程技术获取重组人血小板因子4

Obtaining of human platelet factor 4 by means of genetic engineering technique

目的:用基因工程方法获取重组人血小板因子4(rhPF4),为下一步基础理论研究和临床应用奠定基础。方法:PCR方法获得人PF4编码序列,克隆入质粒pRSET,构建融合表达载体pRSET-PF4,转化大肠杆菌,以IPTG诱导表达融合的人PF4蛋白,经镍柱亲和层析纯化,SDS-PAGE分析所表达的目的蛋白及纯化后蛋白。结果:成功构建了表达载体pRSET-PF4,DNA序列测定结果与预期结果一致。IPTG诱导表达的rh-PF4融合蛋白部分以可溶形式存在,占菌体总蛋白量的11%。亲和层析纯化后目的蛋白纯度为84%。结论:获得原核基因工程表达的rhPF4蛋白。

Objective： To obtain human platelet factor 4 （hPF4） by means of genetic engineering technique. Methods：DNA coding sequence for hPF4 was obtained by PCR and cloned into plasmid pRSET to construct fusion expressed vector pRSET- PF4. After transformed pRSET -PF4 to E. coli DH5α, the bacteria were induced by IPTG. The expressed hPF4 fused protein was purified by Ni - NTA affinity chromatography and identified by SDS - PAGE. Results ： The DNA sequencing showed that the expression vector pRSET - PF4 was constructed successfully. After induced by IPTG, the target protein accounted for 11% of the total bacterial protein and partly was in a soluble form. The purity of the fused protein was up to 84% after Ni - NTA affinity chromatography. Conclusion：The recombinant hPF4 fused protein is obtained.