| 捻转血矛线虫Hc38重组蛋白的表达纯化及抗原性鉴定 |
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| 引用本文： | 何延华,王新华,薄新文,陈南颖,张兴亚,康立超.捻转血矛线虫Hc38重组蛋白的表达纯化及抗原性鉴定[J].新疆农业科学,2011,48(4):750-754. |
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| 作者姓名： | 何延华 王新华 薄新文 陈南颖 张兴亚 康立超 |
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| 作者单位： | 新疆农垦科学院,新疆兵团绵羊繁育生物技术重点实验室,新疆石河子,832000 |
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| 基金项目： | 国家自然科学基金，新疆农垦科学院青年基金 |
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| 摘 要： | 【目的】通过表达和纯化捻转血矛线虫新功能基因Hc38最大开放阅读框ORF1,确定完整的Hc38蛋白天然表达形式、分子量大小和免疫学活性鉴定,为研究该基因功能和基因工程应用奠定基础。【方法】参照本实验室在GenBank上登录的捻转血矛线虫ZJ株的Hc38基因序列(AY594263)设计引物,PCR扩增目的基因并克隆到表达载体中,重组表达载体转化大肠杆菌后IPTG诱导表达,SDS-PAGE和薄层扫描检测分析表达产物,通过亲和层析柱对重组蛋白进行纯化,并用Western bloting和ELISA方法检测其抗原性。【结果】Hc38基因在大肠杆菌中高效表达,表达的融合蛋白相对分子量约为68 KD,表达量占菌体蛋白总量的30%,亲和层析柱纯化出目的蛋白,并能被捻转血矛线虫病绵羊阳性血清和重组表达产物免疫小鼠血清识别,具有反应原性。【结论】实现了Hc38基因的原核表达和纯化,验证了Hc38基因在虫体内的表达形式和抗原性。
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| 关 键 词： | 捻转血矛线虫 Hc38基因 表达 |
Expression Purification and Antigenic Identification of Recombinant He38 Protein of Haemonchus contortus |
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| HE Yan-hua,WANG Xin-hua,BO Xing-wen,CHEN Nan-ying,ZHANG Xin-ya,KANG Li-chao.Expression Purification and Antigenic Identification of Recombinant He38 Protein of Haemonchus contortus[J].Xinjiang Agricultural Sciences,2011,48(4):750-754. |
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| Authors: | HE Yan-hua WANG Xin-hua BO Xing-wen CHEN Nan-ying ZHANG Xin-ya KANG Li-chao |
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| Institution: | HE Yan-hua,WANG Xin-hua,BO Xing-wen,CHEN Nan-ying,ZHANG Xin-ya,KANG Li-chao(Key Laboratory of Sheep Reproduction & Breeding Biotechology,Xinjiang Production and Construction Grops,Shihhottze Xinjiang 832000,China) |
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| Abstract: | 【Objective】To express,purify and characterize recombinant protein of Haemonchus contortus Hc38 gene.【Method】Hc38 gene mostly ORF1 was cloned by RT-PCR with a set of primers designed on complete cDNA(AY594283) with the total RNA extracted from H.contortus adults.After cloning and sub-cloned into pET28a and transformed into E.coli Rosetta(DE3).Recombinant expression Escherichia coli was induced by IPTG,SDS-PAGE、Western-bolt were employed for analyzing the recombinant protein.【Result】pET28a-Hc38 recombinant protein was expressed successfully and its relative molecular weight of expressed fusion protein was about 68 KD,accounting for 30% of total bacteria protein,and could be recognized by positive sera against H.contortus.【Conclusion】Hc38 recombinant protein was expressed and purified successfully.In addition,its size in H.contortus adults and antigenicity were identified by western bolt. |
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| Keywords: | Haemonchus contortus Hc38 gene expression |
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