肝癌相关抗原HAb18G的高效原核表达及其体外功能的研究

High expression of hepatoma associated antigen HAb18G in prokaryotic cells and study on its function in vitro

目的:构建HAb18G的原核表达载体,并在大肠杆菌中诱导高效表达,进行功能、免疫原性测定。方法:采用PCR技术,从已构建的pBluescript/HAb18G克隆载体中扩增HAb18G全长cDNA,克隆入原核表达载体pRSET-C,转化大肠杆菌BL21(DE3)并诱导表达,表达产物用SDS-PAGE检测,表达量用激光薄层扫描检测。纯化表达的HAb18G蛋白,并用明胶酶谱和ELISA法进行测定。结果:双酶切及DNA测序证实,载体构建正确。SDS-PAGE鉴定表明,表达产物的相对分子质量(Mr)为34600,与理论值相符。激光薄层扫描检测,表达量占菌体总蛋白量的33%。明胶酶谱结果呈阴性,但ELISA测定结果呈阳性。结论:实现了HAb18G的原核高效表达。初步证明,该蛋白具有免疫原性但无刺激产生基质金属蛋白酶的功能,为HAb18G蛋白的大量制备及深入研究奠定了基础。

AIM： To construct prokaryotic expression vector of HAb18G, and express high level of this fusion protein in E. coil and to identify its function and immunogenicity. METHODS： The HAblSG full length cDNA from pBlue- script/HAbl8G was obtained by PCR and cloned into pro- karyotic expression vector pRSET-C and then transformed into E. coil 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. RESULTS： 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 （Mr） of the expressed product HAb18G fusion protein was 34 500, which was in accordance with predicted M, 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. CONCLUSION ： 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 HAbI8G protein in great quantities and proceeding to its relative research.