大鼠肝脏F蛋白基因的克隆和表达

目的获得大鼠肝脏F蛋白基因克隆(F-protein’s cDNA);利用原核(大肠杆菌)表达系统表达大鼠肝脏F蛋白。方法提取大鼠肝脏总RNA,对其进行反转录和PCR(RT-PCR)扩增出目的基因(F-protein’s cDNA),然后将其与pUCm-T载体进行连接获得克隆质粒pUCm-T-F并转化到大肠杆菌DH5α中;将测序结果正确的克隆片断重新与表达载体pET-15b连接,获得F抗原表达质粒pET15b-F并将其转化到表达菌株BL21(DE3)pLysS中,用1mmol/L IPTG诱导其表达。结果RT-PCR扩增出的目的基因(F-protein’s cDNA),经测序证明与Gene-bank上提供的F蛋白cDNA序列完全一致;表达后的全菌蛋白进行SDS-PAGE电泳检测,目的蛋白分子量大小约为43 kD,与预期值相符,表达量可达全菌总蛋白量的40%。结论表达的目的蛋白经His-tag柱进行亲和层析纯化,SDS-PAGE检测得到了不含其他杂蛋白的单一F蛋白条带,与豚鼠抗人F蛋白抗血清反应成阳性,说明我们经基因工程方法得到的纯化的F蛋白有免疫活性。

Gene cloning and expression of rat liver-specific F protein

Objective To obtain the clone of rat liver-specific F protein,and to express F protein by prokaryotic expression system.Methods Rat liver total RNA was isolated and first-strand cDNA was reversely transcribed by using the PCR reverse primer.Then the cDNA of F protein was ligated into the clone vector pUCm-T.The segment of F protein's cDNA was subcloned into the expression vector pET-15b,then transformed into E.coli BL21(DE3)pLysS.IPTG was used to induce the target protein to express.Results The cDNA clone was turned to be identical to the sequence published by Gene-bank;SDS-PAGE revealed that the F protein expression product had a Mr43kD and the quantity of the foreign protein expressed by host cell was accounted to be 40 percent of the whole proteins.Conclusion SDS-PAGE reveals the purified protein appears as monoband.Western-blot displays the purified F protien has immunological activity with human-specific guinea pig anti-F protein polyclonal antisera.Thus,F protien is obtained by gene engineering from rat.