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新城疫病毒F48E9株V基因的表达
及其抗血清的制备
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韩放1, 2， 闫丽辉1
，曹殿军1 ，刘培欣1 ，李景鹏2
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（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150001；
2.东北农业大学，黑龙江 哈尔滨150030）
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摘要：
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采用PCR方法从鸡源新城疫病毒（NDV）强毒F48E9 株P基因重组质粒中扩增出了V基因全长cDNA，并引进相应的突变位点，将其克隆到pMD18-T载体，然后亚克隆到原核表达载体pET-30c上，重组质粒经酶切、PCR鉴定及测序，筛选出阳性重组质粒，并命名为pET-30c-V。将pET-30c-V转化大肠埃希氏菌BL21（DE3），用1mmol/L IPTG 37℃过夜诱导表达，SDS-PAGE及Western-blotting分析结果表明，所表达的NDV V重组蛋白主要以包涵体形式存在，分子质量约28ku，与理论值大小相符。将包涵体用8mol/L脲变性，经Ni-NTA柱纯化，透析复性，得到纯化的V蛋白。用复性后的V蛋白免疫21日龄SPF鸡（300μg/只），成功制备了抗鸡NDV V蛋白的抗血清。 |
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关键词：
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新城疫病毒；V基因；原核表达 |
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中图分类号：S 852.659.5：Q 786 文献标识码：
A 文章编号：1673-4696(2006)05-0357-05 |
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Expression of V gene of Newcastle disease virus F48E9 strain and
the preparation of specific antiserum against V protein |
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HAN Fang1, 2, YAN
Li-hui1, CAO Dian-jun1, LIU
Pei-xin1,LI Jing-peng2 |
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(1.National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese
Academy of Agricultural Sciences, Harbin 150001, China; 2.Northeast Agricultural University, Harbin 150030, China ) |
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Abstract: The full length cDNA of NDV V protein gene was cloned from the plasmid containing NDV F48E9 P gene and a necessary mutation for the expression of the V protein gene was introduced by PCR. The mutant V gene was subcloned into the prokaryotic vector pET-30c. After analysis with restriction enzyme digestion, PCR identification and sequencing of the recombinant plasmid, the positive recombinant plasmid was selected and designated as pET-30c-V. The pET-30c-V was transformed into E.coli BL21, and then induced with IPTG at 37℃ overnight. SDS-PAGE and Western-blotting analysis showed that the expressed recombinant V protein presented in inclusion bodies and was 28ku in molecular weight. To prepare specific antiserum against V protein, the recombinant V protein which was purified with Ni-NTA agarose was refolded by dialysis against PBS and water. The antiserum against V protein was generated by immunizing 21 day-old SPF chickens with the purified V protein. |
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Key words: Newcastle disease virus；V gene；prokaryotic expression |
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