Expression of the VP2 Gene Fragment of Goose Parvovirus in Prokaryotic System and Preparation of its Antiserum

HOU Qiu-lian; WANG Jing; LIU Sheng-wang; KONG Xian-wang; HAN Zong-xi; RAN Duo-liang

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August 2005

HOU Qiu-lian, WANG Jing, LIU Sheng-wang, KONG Xian-wang, HAN Zong-xi and RAN Duo-liang. Expression of the VP2 Gene Fragment of Goose Parvovirus in Prokaryotic System and Preparation of its Antiserum[J]. Virologica Sinica, 2005, 20(4): 383-387.

Citation: HOU Qiu-lian, WANG Jing, LIU Sheng-wang, KONG Xian-wang, HAN Zong-xi, RAN Duo-liang. Expression of the VP2 Gene Fragment of Goose Parvovirus in Prokaryotic System and Preparation of its Antiserum .VIROLOGICA SINICA, 2005, 20(4) : 383-387.

鹅细小病毒vp2基因片段在原核系统中的表达及多克隆抗体的制备

侯秋莲 ^1,2 ,
王静 ¹ ,
刘胜旺 ¹ ,
孔宪刚 ^1* ,
韩宗玺 ¹ ,
冉多良 ²

1.
1.中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室黑龙江哈尔滨 2.新疆农业大学动物医学学院新疆乌鲁木齐

摘要

根据鹅细小病毒(Gooseparvovirus,GPV)中国分离株HG5/82基因序列,设计引物,利用PCR技术扩增出HG5/82株vp2基因,将其克隆到pMD18-T载体后,转化入感受态细胞TG1中增殖。筛选阳性质粒,将其与原核表达载体pPROEXTMHTb分别用NcoI酶切后回收目的片断,进行定向连接,产物转化入感受态DH5α,重组质粒经酶切和测序证实目的基因正确克隆到表达载体的预期位点且插入方向正确,构建了含有HG5/82主要结构基因vp25’端969bp片段的原核表达载体。经IPTG诱导后表达出与预期大小相符的约36kDa的融合蛋白,表达形式为包涵体。薄层扫描结果表明表达产物约占菌体总蛋白的21.4%。包涵体通过6mol/L盐酸胍裂解后,利用镍离子亲和树脂进行纯化,用纯化的分子量为36kDa的融合蛋白免疫新西兰白兔,制备兔抗鹅细小病毒部分结构蛋白多克隆抗体。Westernblot分析表明该多克隆抗体与HG5/82毒株具有反应性,说明该融合蛋白具有抗原性。
- 鹅细小病毒
- , vp2基因
- , 克隆
- , 表达
- , 多克隆抗体

Expression of the VP2 Gene Fragment of Goose Parvovirus in Prokaryotic System and Preparation of its Antiserum

Abstract

A 969bp fragment at the 5′- end of the vp2 gene of Goose parvovirus isolate HG5/82 was subcloned into the Nco I site of prokaryotic expression vector pPROEX~{TM}HTb. The recombinant plasmid was transformed into E.coli DH5α and induced with IPTG. SDS-PAGE analysis showed an induced product band about 36kDa, which was corresponding to the size of the fragment. The amount of the recombiant protein was evaluated by densitometric scanning. It indicated that the product was 21.4% of total bacterial protein.The induced bacteria was solubilized by 6mol/L Guanidine hydrochloric acid and purified by ProBond~{TM} Resin. The antiserum against the recombinant protein was obtained by injecting the rabbit with fusion protein. We successfully expressed and purified the fusion protein from E.coli and obtained the antiserum against it, and laid a foundation for future studies on the bioactivity of GPV.
- Goose parvovirus
- , vp2 gene
- , Cloning
- , Expression
- , Polyclonal antibody

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1.
沈阳化工大学材料科学与工程学院沈阳 110142

Expression of the VP2 Gene Fragment of Goose Parvovirus in Prokaryotic System and Preparation of its Antiserum

Abstract: A 969bp fragment at the 5′- end of the vp2 gene of Goose parvovirus isolate HG5/82 was subcloned into the Nco I site of prokaryotic expression vector pPROEX~{TM}HTb. The recombinant plasmid was transformed into E.coli DH5α and induced with IPTG. SDS-PAGE analysis showed an induced product band about 36kDa, which was corresponding to the size of the fragment. The amount of the recombiant protein was evaluated by densitometric scanning. It indicated that the product was 21.4% of total bacterial protein.The induced bacteria was solubilized by 6mol/L Guanidine hydrochloric acid and purified by ProBond~{TM} Resin. The antiserum against the recombinant protein was obtained by injecting the rabbit with fusion protein. We successfully expressed and purified the fusion protein from E.coli and obtained the antiserum against it, and laid a foundation for future studies on the bioactivity of GPV.