学术
Ⅱ型猪圆环病毒ORP2基因的原核表达与初步应用
吴国平[1] 郭川[1] 刘冰心[2] 梁银龙[2] 苏文金[1,2] 曹敏杰[2]
[1]厦门大学生命科学学院,福建厦门361005 [2]集美大学生物工程学院,福建厦门361021
摘 要:
Ⅱ型猪圆环病毒（PCV2）是引起断奶仔猪多系统衰竭综合症（PMWS）的重要病原．本研究用PCR以构建的PCV2 ORF2重组质粒pBS-T-ORP2为模板，扩增截短的ORP2（tORF2）基因亚克隆至表达载体pGEX-4T-3，构建重组表达质粒pGEX-tORP2并转化大肠杆菌BL21（DE3）．经SDS-PAGE及Western blot鉴定，成功表达了谷胱甘肽S转移酶（GST）融合的tORP2，重组蛋白分子量约为45ku，表达量约为菌体总蛋白的20％，重组蛋白具有良好的免疫学活性．用tORP2重组蛋白对20份临床猪繁殖与呼吸障碍综合征（PRRS）阳性猪血清进行Western blot检测，有4份（20％）血清显示PCV2抗体阳性，说明PCV2与PRRSV存在共同感染现象．本研究初步证明表达的tORV2重组融合蛋白可以应用于临床检测．[著者文摘]
文章出处:
《厦门大学学报：自然科学版》-2006年45卷B05期 -129-133页
栏目信息:
分 类 号:
文献标识码:
A
文章编号:
0438-0479（2006）S-0129-05
Prokaryotic Expression and Diagnostic Application of ORF2 of Porcine Circovirus Type Ⅱ
WU Guo-ping, GUO Chuan, LIU Bing-xin, LIANG Yin-long, SU Wen-jin, CAO Min-jie （1. School of Life Sciences, Xiamen University, Xiamen 361005, China; 2. School of Biotechnology, Jimei University, Xiamen 361021, China）
Abstract:
Porcine circovirus type Ⅱ （PCV2） plays a crucial role in the pathogenesis of post-weaning multisystemic wasting syndrome （PMWS） in swine. The ORF2 gene encoding the capsid protein of PCV2 was amplified by PCR and cloned into plasmid pBS-T to construct recombinant plasmid pBS-T-ORF2, which was used as template to amplify the truncated ORF2 （tORF2）. The tORF2 gene was consequently cloned into the expression vector of pGEX-4T-3 for prokaryotic expression in E. coli. Expression level of the recombinant GST-tagged tORF2 protein reached approximately 20% of the total bacterial proteins and its molecular weight was approximately 45 ku identified by SDS-PAGE and Western blot. The recombinant protein was used as antigen to establish a Western blot diagnostic assay. Twenty PRRS positive swine serum samples were detected by this Western blot assay. The positive rate of these sera was 20% （4/20）, indicating that coinfection of PRRSV and PCV2 exist in swine. Taken together, the recombinant tORF2 protein reporteded in the present study may play a critical role in the detection of PCV2 antibodies immunologically.[著者文摘]
Key words:
porcine cireovirus type Ⅱ; ORP2 gene; prokaryofic expression; diagnostic application
基金资助:
福建省科技攻关计划重点项目（2003N083）和厦门市科技攻关计划重点项目（3502Z20031054）资助
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