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猪生殖与呼吸综合征病毒多基因重组
真核表达质粒的构建及诱导的小鼠体液免疫
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孙新峰，张淼涛*
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（西北农林科技大学 动物医学院，陕西 杨凌712100）
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摘要：
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根据猪生殖与呼吸综合征病毒已知序列设计了3对引物，采用RT-PCR方法分别扩增ORF3、ORF5和ORF6基因，并将其依次与真核表达质粒pcDNA4.0定向连接，构建了重组质粒pcDNA4.0-ORF6-ORF3-ORF5，经提纯后，给试验组小鼠后肢胫前肌注射，每只100μg，2周后再注射1次。首免后每隔7 d采血1次，用间接ELISA检测血清抗体水平并测定其中和抗体水平。结果显示，所构建的重组质粒能够诱导小鼠产生很高的ELISA抗体和中和抗体水平。 |
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关键词：
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猪生殖与呼吸综合征病毒；ORF3；ORF5；ORF6；真核表达质粒；体液免疫 |
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中图分类号：S 852.659.6：Q 782
文献标识码：
A 文章编号：1673-4696(2008)03-0209-05 |
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Construction of recombinant plasmid pcDNA4.0-ORF6-ORF3-ORF5 of
PRRSV and humoral immunity induced by the plasmid in mice |
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SUN Xin-feng,ZHANG Miao-tao
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(College of Veterinary Medicine,Northwest Sci-Tech University of Agriculture and Forestry,Yangling 712100,China) |
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Abstract: Based on the published nucleotide sequence of porcine reproductive and respiratory syndrome virus(PRRSV),three pairs of primers were designed and used to amplify ORF3,ORF5 and ORF6 genes by RT-PCR,and the amplified products were cloned into eukaryotic expression plasmid pcDNA4.0 to construct recombinant plasmid pcDNA4.0-ORF6-ORF3-ORF5.Then,10 Kunming mice were inoculated with the purified pcDNA4.0-ORF6-ORF3-ORF5 into hind legs tibial muscle(50μg per leg) by intramuscular injection,and were boosted on week 2 post-inoculation.The levels of ELISA antibodies and neutralizing antibodies were detected in peripheral bloods of the inoculated mice or the boosted mice every 7 days post-inoculation or post-booster-inoculation by indirect ELISA.The results showed that the expressed envelope protein from the plasmid could induce to produce high levels of ELISA antibodies and neutralizing antibodies. |
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Key words: PRRSV;ORF3;ORF5;ORF6;eukaryotic expression plasmid;humoral immunity |
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