摘要
目的克隆简单异尖线虫L-样半胱氨酸蛋白酶基因(AsCP)全长,研究其表达特性。方法根据GenBank中简单异尖线虫表达序列标签L-样半胱氨酸蛋白酶基因的部分信息,设计特异引物,用cDNA末端快速扩增技术扩增3′端部分,获得基因全长序列。根据基因全长序列设计引物,以简单异尖线虫总RNA为模板,RT-PCR扩增AsCP基因编码序列,产物经EcoRⅠ和SalⅠ双酶切,克隆至表达载体pET32а(+),转化大肠埃希菌BL21(DE3)株,以异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,表达效果经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测。结果3′端扩增片段大小为1211bp,拼接完整后基因全长1462bp,编码411个氨基酸,与秀丽隐杆线虫的L-半胱氨酸蛋白酶相似性达36.4%;重组载体pET32a(+)-AsCP经EcoRⅠ和SalⅠ双酶切后有一条约1150bp的条带,测序结果显示重组载体构建成功。SDS-PAGE结果表明,重组蛋白相对分子质量约为Mr60000(含6个组氨酸的标签),与目的蛋白相符。用不同浓度的IPTG诱导对表达量的影响很小,1mmol/LIPTG诱导2h后表达量达到最高水平。结论成功克隆并表达了L-样半胱氨酸蛋白酶。
Objective To clone and express the full lenth of L-like cysteine protease gene of Anisakis simplex （AsCP） . Methods According to L-like cysteine protease encoding gene of A. simplex from GenBank EST database, specific primers were designed to amplify 3′-end of AsCP gene using rapid-amplification of cDNA ends （RACE）, and the full lenth of the L-like cysteine protease gene was obtained. Specific primers were designed according to the full length of the gene. Using total RNA of A. simplex third-stage larvae, coding sequence of the AsCP gene was amplified by RT- PCR. The PCR product was digested by EcoR I and Sal I, and cloned into pET32а（＋） vector. The recombinant plasmid was checked by double enzyme digestion and sequencing, and the positive recombinant plasmid was transformed into Escherichia coli BL21（DE3）. Expression of the protein induced by IPTG of gradient concentration （0.2, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mmol/L） and by the same concentration （1 mmol/L） of IPTG at different time（0, 1, 1.5, 2, 2.5, 3, 3.5, and 4 h） was conducted. The expression situation of recombinant protein was analyzed by SDS-PAGE. Results A 1 211 bp of 3′-end of AsCP gene was amplified by 3′RACE, full length of the gene was 1 462 bp and coding 411 amino acids. It showed 36.4% identity with the L-cysteine protease of Caenorhabditis elegans. Double enzyme digestion of the constructed recombinant plasimid pET32a（＋）-AsCP showed that there was about 1 150 bp fragment, the constructed recom- binant plasmid was then identified by sequencing. SDS-PAGE showed that the recombinant protein （Mr 60 000） was identical with the target. IPTG showed little effect on the protein expression, and the production of protein was up to maximum after 2 hours induction. Conclusion The AsCP gene has been cloned and expressed.
出处
《中国寄生虫学与寄生虫病杂志》
CAS
CSCD
北大核心
2010年第2期135-138,共4页
Chinese Journal of Parasitology and Parasitic Diseases
基金
福建省科技计划项目(No.2008N2005)~~
