人PDGF-A基因的克隆、表达及表达产物的纯化

Cloning and expression of platelet-derived growth factor-chain A gene and purification of expressed product

目的 克隆血小板衍生的生长因子A链 (PDGF A)的基因 ,并在大肠杆菌中表达。方法 利用RT PCR法 ,从人肝癌细胞系 (HHCC)的总RNA中 ,扩增PDGF A的全长cD NA ,再用PCR法扩增PDGF A成熟蛋白的全长编码序列。编码序列经测序验证后 ,克隆入表达载体pGEX 4T 1中 ,构建PDGF A的原核表达载体 ,在大肠杆菌中诱导表达目的蛋白 ,并进行谷胱甘肽 (GST)亲和层析纯化。结果 以RT PCR扩增的PDGF A基因的全长为 12 5 5bp ,以PCR扩增得到其成熟蛋白的编码序列为 5 31bp。构建了PDGF A基因的原核高效表达载体pGEX PDGF A ,表达产物主要位于包涵体内。对包涵体蛋白进行变性和复性处理后 ,利用亲和层析法获得纯化的目的蛋白。结论 成功地扩增到PDGF A成熟蛋白的编码序列 ,并在大肠杆菌高效表达 ,为进一步对PDGF

Aim To clone the gene of A chain of human platelet derived growth factor (PDGF A) and express the protein in E. coli . Methods Full length cDNA of PDGF A was amplified by RT PCR from the total RNA of human hepatocarcinoma cell line (HHCC). DNA sequence encoding the PDGF A mature protein was amplified by PCR. The gene encoding PDGF A mature protein being proved to be right by sequencing was cloned into expression vector pGEX 4T 1 to construct recombinat expression vector pGEX PDGF A and the protein was expressed in E. coli. The expressed protein was purified by GST affinity chromatography.Results The full length sequence of PDGF A cDNA was 1 255 bp and the encoding DNA sequence of mature protein was 531 bp. The high efficient expression vector pGEX PDGF A was constructed. The expressed product was mainly located in inclusion body. After the treatment of denaturation and renaturation of inclusion body, the purified targeted protein was obtained by affinity chromatography. Conclusions The gene encoding mature PDGF A protein has been amplified successfully and was expressed high efficiently in E. coli . The results lay the foundation for further study on function of PDGF A .