构建柯萨奇B4病毒非结构蛋白P2C与质粒PQE-4．0的重组质粒

Constructing recombinant plasmid of nonstructural protein P_2C of coxsackie virus B_4 virus and plasmid PQE-4.0

背景:研究表明柯萨奇B4病毒感染与1型糖尿病发病有关,但目前尚未明确其关系的确切机制。目的:构建柯萨奇B4病毒非结构蛋白P2C与质粒PQE-4.0的重组质粒,转入大肠杆菌中表达,并对表达产物进行功能鉴定。设计、时间及地点:随机对照实验,于2007-03/08在吉林大学免疫学教研室完成。材料:CVB4、HeLa细胞病毒均为吉林大学病原生物教研室保存;菌株E.coli DH5α与M15为东北师范大学遗传所保存;质粒PUCm-T载体、PQE4.0载体由东北师范大学曾宪录教授惠赠。方法:提取柯萨奇B4病毒总RNA,经反转录-聚合酶链反应扩增非结构蛋白P2C基因,与PUCm-T载体连接,将PUCm-T-P2C质粒进行BamHⅠ和HindⅢ双酶切,回收目的片断并定向克隆到PQE4.0表达载体中酶切鉴定。将PQE4.0-P2C阳性重组质粒转化大肠杆菌M15,在IPTG诱导下表达。主要观察指标:①以十二烷基硫酸钠-聚丙烯酰胺凝胶电泳检测柯萨奇B4病毒非结构蛋白P2C的表达。②采用淋巴细胞转化实验和酶联免疫吸附实验检测大肠杆菌M15细胞重组蛋白粗提物。结果:①反转录-聚合酶链反应扩增得到的987bp的cDNA片段,克隆入PUCm-T载体,酶切释放出长度约2773bp和987bp的两条片段,其中一条与非结构蛋白P2C的聚合酶链反应扩增产物片段大小基本一致,序列分析与Genbank报道的序列一致。目的片段克隆入PQE4.0载体,酶切释放出长度约3400bp和987bp的两条片段,其中一条与非结构蛋白P2C的聚合酶链反应扩增产物片段大小基本一致,获得PQE4.0-P2C重组表达质粒。②十二烷基硫酸钠-聚丙烯酰胺凝胶电泳结果显示未诱导组无明显蛋白带,而诱导组在31000位置均出现较明显的蛋白带,且随着诱导时间延长蛋白表达量也随之增加。③淋巴细胞转化实验与酶联接免疫吸附试验测定结果表明,表达产物具有良好的抗原性与特异性。结论:柯萨奇B4病毒非结构蛋白P2C在大肠杆菌中高效表达,且表达产物具有一定的免疫活性。

BACKGROUND： Coxsackie virus B4 （CVB4） is associated with type 1 diabetes mellitus, but there is no defined mechanism of their correlation. OBJECTIVE： To construct recombinant plasmid of CVB4 non-structural protein P2C and plasmid PQE4.0, then transfer it into E coli and determine the function of expression product. DESIGN, TIME AND SETTING： A randomized control experiment was performed at the Department of Immunology of Jilin University from March to August 2007. MATERIALS： HeLa cell and CVB4 were preserved at the Department of Aetiology of Jilin University. Strain E.coli DH5α and M 15 were preserved at the Heredity Research Institute of Northeast Normal University. Plasmid PUCm-T and PQE 4.0 were donated by professor Zeng from Northeast Normal University. METHODS： CVB4 total RNA was extracted. A full-length cDNA of nonstructural protein P2C gene was obtained using reverse transcription-polymerase chain reaction （RT-PCR） amplification of total RNA extracted from virus. The RT-PCR product was cloned into PUCm-T vector. PUCm-T-P2C plasmid was identified with BamH Ⅰ and Hind Ⅲ and oriented to subclone into PQE4.0 expression vector transfected E coli M15 to screen the positive clone and to identify the recombinant plasmid by double enzyme digestion. The target protein was induced by isopropy- β -D- thiogalactoside （IPTG）. MAIN OUTCOME MEASURES： CVB4 nonstructural protein P2C was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）. E.coli M15 recombination protein crude extract was assayed by lymphocyte transformation and enzyme-linked immunosorbent assay （ELISA）. RESULTS： A cDNA fragment of 987 bp was obtained by PCR, then cloned into PUCm-T vector and obtained two fragments of 2 773 bp and-987 bp. One of them matched with PCR products of nonstructural protein P2C and its sequence was in conformity with the sequence being published in Genebank. The target fragment was cloned into PQE4.0 vectors and obtained two fragments of 3 400 bp and 987 bp, with digestion which paired with PCR products of nonstructural protein P2C. The recombinant plasmid PQE 4.0-P2C was successfully constructed. The expression of P2C protein by SDS-PAGE with a molecular weight of 31 kDa in the induction group and the protein content increased along with the inducing time. But in non-induction group there was no evident lane of the protein. Lymphocyte transformation and ELISA showed that expression products had favorable antigenicity and specificity. CONCLUSION： CVB4 nonstructural protein P2C expresses in Ecoli with high performance and expression product has immunologicalactivity to some extent.