摘要
目的构建包含人前列腺干细胞抗原(PSCA)的原核表达质粒,并进行诱导表达、蛋白纯化及鉴定。方法通过PCR扩增人PSCA基因片段,与过渡载体pGEM(-TEasy连接后进行测序鉴定,鉴定正确后,将PSCA基因片段酶切并插入含有谷胱甘肽s-转移本科GST标签的原核表达载体pET42a,得到重组质粒pET42a-PSCA。将pET42a-PSCA转化大肠埃希菌BL21(DE3),在IPTG诱导下表达GST-PSCA融合蛋白,用Glutathione-Sepharose4B柱对融合蛋白进行纯化,纯化产物进行SDS-PAGE及Western blotting鉴定。结果 PSCA基因的PCR产物大小与预期相符,测序结果与GenBank中的PSCA序列一致。pET42a-PSCA原核表达质粒构建正确,SDS-PAGE及Western blotting检测显示融合蛋白诱导表达成功,分子量大小约为43kD。结论成功构建了人PSCA的原核表达质粒,能在BL21菌株中表达,且纯化后得到较高纯度的GST-PSCA融合蛋白,为后续抗前列腺癌基因疫苗研究奠定了基础。
Objective To construct the prokaryotic expression plasmid of human prostate stem cell antigen （PSCA）,to induce the expression of GST-PSCA fusion protein in E. coli BL21,and to identify the purified recombinant fusion protein. Methods The fragment of PSCA gene was amplified by PCR,and then cloned into the pGEM-T Easy vector. The transitional plasmid Teasy-PSCA was identified by DNA sequencing. The PSCA gene was digested from the plasmid Teasy-PSCA by restrictive enzyme BamH I and Sal I,and then inserted into the pET42a vector which contains a glutathione s-transterase （GST） tag. Following the double restriction enzyme digestion,the recombinant plasmid pET42a-PSCA was obtained and transformed into E. coli BL21 （DE3）. The expression of GST-PSCA fusion protein was induced with IPTG. The recombinant fusion protein was purified by passing over a Glutathione Sepharose 4B column,and was identified by SDS-PAGE and Western blotting. Results The length of amplified PSCA gene fragment was consistent with that expected,and the sequence was correct as exemplified by the PSCA gene reported in GenBank. The result of enzyme digestion indicated that the prokaryotic expression plasmid pET42a-PSCA was successfully constructed. After transformation with pET42a-PSCA and induction with IPTG,the recombinant target protein of about 43kD was obtained. The GST-PSCA fusion protein was correctly identified by SDS-PAGE and Western blotting. Conclusions The prokaryotic expression plasmid of human PSCA gene has been successfully constructed. The GST-PSCA fusion protein may express and be purified in E. coli BL21,and it lays a foundation for further study on the anti-prostate cancer gene vaccine.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2010年第8期986-988,共3页
Medical Journal of Chinese People's Liberation Army
基金
国家高技术研究发展计划(2006AA02A237,2007AA02Z451)
国家自然科学基金(30772002)
