【作者】 杨庆贵；

【导师】 李朝品；

【作者基本信息】 安徽理工大学 ， 病原生物学， 2005， 硕士

【摘要】 目的 对淮南地区粉尘螨Ⅰ、Ⅱ类变应原cDNA进行克隆、表达。 方法 采集淮南地区粉尘螨进行人工饲养,用Trizol提取总RNA,以OligodT-Adaptor Primer为反转录引物,使用TaKaRa RNA LA PCR TMKit(AMV)Ver.1.1合成cDNA。根据GenBank粉尘螨Der f1、Der f2 cDNA基因序列(登录号分别为:emb | X65196.1, D10448),分别设计、合成一对特异性引物,采用RT-PCR、PCR法扩增出目的基因片段,按照常规的基因操作技术进行Der f1 cDNA、Der f2 cDNA的克隆、亚克隆、表达及鉴定。挑选出阳性克隆菌(含pMD-18T-Der f1、pMD-18T-Der f2)进行序列测定,并亚克隆至表达质粒,构建重组表达质粒pET32a(+)-Der f1、pET32a(+)-Der f2,诱导Der f1、Der f2基因的表达,进行Der f1、Der f2蛋白的SDS-PAGE分析,并对SDS-PAGE蛋白条带进行凝胶扫描定量分析。 结果 ①行RT-PCR、PCR,自粉尘螨基因组中特异扩增出大小约646bp、455bp基因片段。②pMD-18T-Der f1和pMD-18T-Der f2重组质粒中插入片段的测序结果与GenBank登录的相应序列比较,发现Der f1核苷酸同源性为99.52%,其读码框自9至638;Der f2与郝敏麒等报道的广州地区粉尘螨Der f2的cDNA序列(AF346905)相比,未发现有插入序列。③表达载体pET32a(+)-Der f1、pET32a(+)-Der f2经酶切及PCR扩增后,在预期位置上可见DNA条带,证实表达载体构建成功。④含pET32a(+)-Der f1、pET32a(+)-Der f2工程菌经超声破碎集菌后,分别取其上清及沉淀行SDS-PAGE分析,发现沉淀中有目的蛋白的表达,其分子量分别约为45kDa、34kDa。 结论 ①成功进行粉尘螨Der f1、Der f2 cDNA体外扩增,序列分析结果证实,淮南地区Der f1 cDNA与国外粉尘螨Der f1 cDNA序列存在一定差异,Der f2 cDNA与国内粉尘螨Der f2 cDNA存在一定差异,主要表现为缺失一约87bp的插入序列。②成功地构建了Der f1、Der f2 cDNA的重组表达质粒pET32a(+)-Der f1、pET32a(+)-Der f2,在大肠杆菌中高效表达,获得大量rDer f1和rDer f2,为螨性变态反应性疾病特异性免疫诊断和治疗试剂盒的研制奠定了坚实的实验基础。更多还原

【Abstract】 Objective To construct and express prokaryotic expression plasmid of the cDNA coding for group 1 and group 2 allergen of Dermatophagoides farinae which collected in Huainan area.Methods Firstly, the Dermatophagoides mites were collected, identified and cultured in our laboratory. Secondly, the total RNA of D. farinae was extracted with the Trizol reagent. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to acquire cDNA. Then PCR was performed in a 50ul reaction mixture in an automatic thermal cycler, and the PCR product was visualized under UV light and photographed. After purified, the target fragments were cloned into the compatible sites of the T-vector pMD-18T, the recombinant plasmid pMD-18T-Der f 1, pMD-18T-Der f 2 were digested with restriction endonuclease and were subcloned into expression vector pET32a (+), respectively. The positive recombinants were transferred into E.coli BL21 and identified by restriction endonuclease digestion and PCR. Finally, the genetically engineered bacteria including pET32a (+)-Der f 1 and pET32a (+)-Der f 2 plasmids were induced by IPTG, and the expression was analyzed with the methods of SDS-PAGE and gel densitometic scanning. Results The results revealed that the cDNA of Der f 1 and Der f 2 were specifically amplified from total RNA of Dermatophagoides farinae by RT-PCR and PCR, recombinant plasmid pMD-18T-Der f 1, pMD-18T-Der f 2 were successfully digested by BamH I /Sac I , Sac I /Not I , and the products of digestion were identical with the predicted one, respectively. Sequence analysis also showed that the cDNA Der f 1 was 99.52% affinity in comparison with DNA sequence published on GenBank (emb | X65196.1), and insertion sequence wasn’t be found in cDNA Der f 2, while comparison with other cDNA sequence (locus: AF346905). The prokaryotic expression plasmids pET32a (+)-Der f 1 and pET32a (+)-Der f 2 could express a specific 45kDa and 34 kDa protein, which accoutered for about 15% and 16% of the total protein of recombinant bacterial, in E.coli BL21, respectively. Conclusion The sequence of Der f 1 cDNA and Der f 2 cDNA were different in various fauna, respectively. The prokaryotic expression pl;,smids, which eontair. Der f 1 and Der f 2 cDNA from Huainan area, have been constructed successfully and can express objective protein in E.coli DL21.更多还原