Xu Peng, Dou Ke-feng, Chen Zhi-nan, Xing Jin-liang, Li Yu, Kong Ling-en
Department of Hepato-Biliary Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2005 Nov;21(6):734-7.
To construct prokaryotic expression vector of HAb18G, and express high level of this fusion protein in E. coli and to identify its function and immunogenicity.
The HAb18G full length cDNA from pBluescript/HAb18G was obtained by PCR and cloned into prokaryotic expression vector pRSET-C and then transformed into E. coli BL21(DE3) to induce its expression. Expressed products were analyzed by SDS-PAGE and laser thin layer scan. The purified HAb18G protein was identified by gelatin enzymogram and ELISA.
Endonuclease digestion and DNA sequencing proved that HAb18G cDNA was cloned correctly into the expression vector. Result of SDS-PAGE showed that the relative molecular mass of the expressed product HAb18G fusion protein was 34,600, which was in accordance with predicted relative molecular mass value. Laser thin layer scan showed that the expressed product accounted for 33% of the total bacteria protein. Result of enzymogram was negative whereas the result of ELISA was positive.
It was testified that the protein HAb18G has immunogenicity but no bioactivity. The high level prokaryotic expression of HAb18G lay the foundation for manufacturing the HAb18G protein in great quantities and proceeding to its relative research.
构建HAb18G原核表达载体，在大肠杆菌中高效表达该融合蛋白，并鉴定其功能及免疫原性。
采用PCR技术从pBluescript/HAb18G中获取HAb18G全长cDNA，克隆至原核表达载体pRSET-C，转化至大肠杆菌BL21(DE3)诱导表达。表达产物经SDS-PAGE和激光薄层扫描分析。纯化的HAb18G蛋白经明胶酶谱法和ELISA鉴定。
酶切及DNA测序证明HAb18G cDNA正确克隆至表达载体。SDS-PAGE结果显示，表达产物HAb18G融合蛋白相对分子质量为34 600，与预测相对分子质量值相符。激光薄层扫描显示，表达产物占细菌总蛋白的33%。酶谱结果为阴性，而ELISA结果为阳性。
证明HAb18G蛋白具有免疫原性但无生物活性。HAb18G的高效原核表达为大量制备HAb18G蛋白及后续相关研究奠定了基础。
