猪繁殖与呼吸综合征病毒08HuN株全长cDNA克隆的构建
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摘要
猪繁殖与呼吸综合征是由猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus, PRRSV)引起的以母猪繁殖障碍和仔猪呼吸道症状及死亡为主要特性的高度传染性疾病,给养猪业造成了巨大的经济损失。本研究对PRRSV 08HuN株全基因组进行了核苷酸序列测定,将08HuN株基因组序列与其它3个高致病性PRRSV变异株进行比较发现08HuN基因组内有14处氨基酸点突变和42处沉默突变,这些分析为揭示高致病PRRSV毒株的毒力提供了有用的信息。在分析沉默突变时,发现在08HuN株沉默突变中发现了同义密码子突变倾向于从高频密码子向低频密码子突变的现象,在国内外首次提出基因的沉默突变也可能成为PRRSV毒力发生变异的原因,丰富了有关PRRSV遗传变异的理论。本研究还利用反向遗传学技术,构建了PRRSV 08HuN株全长cDNA克隆,并采用酶切连接的方式对全长cDNA克隆中的非同义突变点进行纠正。PRRSV 08HuN株的全长cDNA克隆的构建为进一步获得该毒株的感染性cDNA克隆和深入研究08HuN株的毒力因子以及分子致病机理奠定了基础。
根据猪繁殖与呼吸综合征病毒(PRRSV) 08HuN株基因组6个片段的测序结果,A、E和F片段均有符合下一步克隆要求的正确克隆分子,B、C和D片段则通过酶切连接的方式进行非同义突变的纠正。将A和E片段的正确克隆和B、C和D片段的重组正确克隆均亚克隆至pVAX1载体,F片段的正确克隆亚克隆至ppoly2/sfinot载体。利用各片段之间重叠部分的酶切位点依次拼接成PRRSV 08HuN株全长cDNA分子。
Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of PRRS. It caused highly contagious disease in swine characterized by reproductive disorder in sows and gilts, respiratory diseases and death in piglets, resulting in great economic losses to pig industry. The full-length genome of PRRSV 08HuN strain was sequenced in this article. Comparison with the other three highly pathogenic PRRSV variants revealed 14 point mutations of amine acid and 42 silent mutations in the genome of 08HuN strain, providing useful information on the virulence of highly pathogenic PRRSV strains.In the analysis of silent mutations, we found that there is a codon preference for low-frequency codons to high-frequency codons in the 08HuN strain. To our knowledge, this is the first proposal at home and abroad that silent mutations maybe a contributing factor to the virulence difference of PRRSV virus.The findings also enrich the theory of PRRSV genetic variation. The establishment of full-length cDNA clone of PRRSV 08HuN strain by utilizing reverse genetic manipulation technology. Nonsynonymous mutational sites within full-length cDNA clone were corrected by enzyme digestion and splice. The successful construction of full-length cDNA clone provided fundamental materials for establishment of infectious clone of 08HuN strain and further research on virulence factor and pathogenesis in molecular level of 08HuN strain.
According to the sequencing results of six fragments of the genome of porcine reproductive and respiratory syndrome virus (PRRSV) 08HuN strain,fragment A, E and F all contained correct clone molecules which meet the requirements of further clone, while several nonsynonymous mutational sites within the fragment B, C and D were corrected by enzyme digestion and splice.The correct clone of fragment A, D and the recombinant corrected clone of fragment B, C and D were all subcloned in pVAXl vector. The correct clone of fragment F was subcloned in ppoly2/sfinot vector. PRRSV 08HuN strain full-length cDNA molecule was obtained by enzyme digestion and splice in order using the restriction enzyme cutting sites located in the overlapping region of six fragments.
根据猪繁殖与呼吸综合征病毒(PRRSV) 08HuN株基因组6个片段的测序结果,A、E和F片段均有符合下一步克隆要求的正确克隆分子,B、C和D片段则通过酶切连接的方式进行非同义突变的纠正。将A和E片段的正确克隆和B、C和D片段的重组正确克隆均亚克隆至pVAX1载体,F片段的正确克隆亚克隆至ppoly2/sfinot载体。利用各片段之间重叠部分的酶切位点依次拼接成PRRSV 08HuN株全长cDNA分子。
Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of PRRS. It caused highly contagious disease in swine characterized by reproductive disorder in sows and gilts, respiratory diseases and death in piglets, resulting in great economic losses to pig industry. The full-length genome of PRRSV 08HuN strain was sequenced in this article. Comparison with the other three highly pathogenic PRRSV variants revealed 14 point mutations of amine acid and 42 silent mutations in the genome of 08HuN strain, providing useful information on the virulence of highly pathogenic PRRSV strains.In the analysis of silent mutations, we found that there is a codon preference for low-frequency codons to high-frequency codons in the 08HuN strain. To our knowledge, this is the first proposal at home and abroad that silent mutations maybe a contributing factor to the virulence difference of PRRSV virus.The findings also enrich the theory of PRRSV genetic variation. The establishment of full-length cDNA clone of PRRSV 08HuN strain by utilizing reverse genetic manipulation technology. Nonsynonymous mutational sites within full-length cDNA clone were corrected by enzyme digestion and splice. The successful construction of full-length cDNA clone provided fundamental materials for establishment of infectious clone of 08HuN strain and further research on virulence factor and pathogenesis in molecular level of 08HuN strain.
According to the sequencing results of six fragments of the genome of porcine reproductive and respiratory syndrome virus (PRRSV) 08HuN strain,fragment A, E and F all contained correct clone molecules which meet the requirements of further clone, while several nonsynonymous mutational sites within the fragment B, C and D were corrected by enzyme digestion and splice.The correct clone of fragment A, D and the recombinant corrected clone of fragment B, C and D were all subcloned in pVAXl vector. The correct clone of fragment F was subcloned in ppoly2/sfinot vector. PRRSV 08HuN strain full-length cDNA molecule was obtained by enzyme digestion and splice in order using the restriction enzyme cutting sites located in the overlapping region of six fragments.
引文
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