摘要
应用trizol裂解法提取象山港网箱养殖大黄鱼肝脏总RNA,采用RTPCR获得大黄鱼cDNA;通过设计特异引物进行PCR扩增,将PCR产物转化到质粒并直接测序,得到1.7kb的目的基因;同时将此目的基因连接到表达质粒pSBET中,在大肠杆菌BL21中进行诱导表达以及表达产物的分析。克隆基因的表达产物经SDSPAGE分析表明,HSP基因的蛋白分子量为60kDa左右,与阅读框的编码大小一致;表达蛋白经纯化后免疫小鼠制备抗血清。Westernblotting检测结果表明,制备的抗血清与HSP蛋白起较强的特异性反应。HSP基因的系统进化分析表明,大黄鱼与非洲爪蟾最为接近,从侧面印证了脊椎动物中两栖纲动物与鱼纲动物的亲源关系。通过大黄鱼HSP基因的提取和分析,可为以后制备出核酸探针、筛选和克隆出一批具有优良性状的基因、构建基因文库等研究工作奠定基础。
With the protocol of trizol, total RNA was extracted from the liver tissue of large croaker （Pseudosciaena crocea） cultured in Xiangshan Bay;RT-PCR was used to obtain cDNA of P. crocea. Special primers were designed for HSP amplification, after sequencing and cloning, the HSP gene was ligated into the expression vector pSBET. The recombinant plasmid was transformed into Escherichia coli BL21 and then induced to express by IPTG. The molecular weight of HSP was about 60kDa by SDS-PAGE, consistent with the deduced size. The expressed fusion protein was purified and then was employed to immunize the mice for preparing specific antiserum, which reacted strongly and specifically to HSP by western blotting. The phylogenic analysis showed： HSP amino acid sequence was highly conserved, and nucleotide sequence of P. crocea shared very high homology with Venous leaves （African clawed frog） , which confirmed the hypothesis that the Amphibian was subsequent partially to the Pisces in phylogentics. Phylogenic construction would provide basis for follow-up works including nucleotide probe preparation, clone and selection of some fine functional genes for culture, and building of P. crocea cDNA library. Combining gene with SDS-PAGE would be an effective way for assessing the levels of genetic diversity in fishes respectively from DNA level.
出处
《海洋与湖沼》
CAS
CSCD
北大核心
2006年第4期337-341,共5页
Oceanologia Et Limnologia Sinica
基金
国家高技术研究发展计划("863")项目
2002AA603021号
宁波市重大招标项目
2004C100040号。
关键词
大黄鱼
热激蛋白基因
原核表达
抗血清
Pseudosciaena crocea, Heat shock protein （HSP） gene, Prokaryotic expression, Antiserum
