广西猪繁殖与呼吸综合征病毒的分子生物学研究
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摘要
近来，广西一些养殖场频繁发生以母猪流产、死胎、木乃伊、仔猪呼吸道症状等为特征的流行性传染病，怀疑为猪繁殖与呼吸综合征病毒(PRRSV)所致。本研究应用针对PRRSV的N基因的特异性引物P1／P2，通过RT-PCR技术对从广西南宁市郊区收集的仔猪病料进行检测，结果为阳性。将材料接种于易感细胞—Marc-145细胞，经过6代盲传，结果出现了典型的细胞病理变化(CPE)，与对照株病变相似，经鉴定为PRRSV，成功地分离出广西第一株PRRSV毒株，命名为GXA株，其毒价为10~(5.33)TCID_(50)／ml。
参考VR-2332和OH-1a株基因序列，设计了3对引物E1／E2、M1／M2、N1／N2，分别扩增GXA株的E、M、N基因。并进行克隆和测序。结果分别扩增出大小为668bp、582bp和443bp的目的片段。应用DNAstar生物学软件对所测得的序列进行处理后，得到GXA株的E、M和N基因ORF全长分别为603nt、524nt和372nt，分别编码200、174和123个氨基酸。将此3个基因序列分别与国内外已发表的ATCC VR-2332、MLV、BJ-4和LV等13个毒株相应基因序列进行了同源性比较。结果发现GXA株与美洲型毒株E基因的核苷酸及其推定的氨基酸的同源性分别为88.7-99.7％和93.8-100％，与欧洲型代表株—LV株的核苷酸及其推定的氨基酸的同源性分别为64.7％和66.3％；GXA株与美洲型毒株M基因的核苷酸及其推定的氨基酸的同源性分别为94.5-100％和96.6-100％，与LV株的核苷酸及其推定的氨基酸的同源性分别为69.2％和78.9％；GXA株与美洲型毒株N基因的核
广西大学2004届硕士毕业论文
昔酸及其推定的氨基酸的同源性分别为93.8一1 00%和95.2一1 00%，与LV株的核
昔酸及其推定的氨基酸的同源性分别为66.3%和63.9%。同源性分析显示，GxA
株与VR一2332、劫LV和BJ一4的同源性非常高，而GXA与LV同源性非常低。构建
的系统发育树分析，GXA与VR一2332、MLV及BJ一4株亲缘关系比较密切。从而表
明GXA株属于美洲型毒株，可能来源于疫苗株。
将GXA株的E、M、N基因从重组质粒 pMD一E、pMD一M、pMD一N中亚克隆至原
核表达载体PE丁一32a上，构建了原核表达重组质粒PE丁一E、pE丁一M和PE丁喇，并
通过酶切鉴定，证实构建成功。
In recent, some pig farms in Guangxi occur frequently an infectious disease , which characters are abortion , death of embryo , mummified fetuses , and respiratory problems in piglet . Pathogen of the disease was considered as porcine reproductive and respiratory syndrome virus (PRRSV) . In this study , we identified that the sample collected from the farm in Nanning was positive by RT-PCR with a pair of specific N gene primers of PRRSV . Based on it, permissive cells , Marc-145 cells were inoculated with the sample . After six blind passages , typical cytopathogenic effect (CPE) was observed on cells as well as the positive control .And PRRSV was positive in the cells and culture medium by RT-PCR . The first PRRSV strain in Guangxi was isolated successfully, named GXA strain . And its infection titer was 105.33 TCID50/ml.
According to VR-2332 and CH-1a strain , designed three pairs of primers E1/E2 , M1/M2 , N1/N2 ,which amplified the fragments of GXA strain's E , M , N gene , respectively . The amplified fragments were cloned and sequenced . The length of E , M and N gene fragment is 668 ,582 and 443 base pairs (bps), respectively . Processed the sequences by DNAstar software , obtained the length of E , M and N gene sequence is 603 ,524 and 372 nucleotides(nts), encoding 200 , 174 and 123 amino acids .respectively . Comparison of the homology among GXA , VR-2332 , MLV , BJ-4 , Lelystad virus (LV) and other 9 strains of PRRSV , the results shown that the homology of nucleotide sequences and deduced amino acid sequences of E gene among GXA and Norther American strains was 88.7-99.7% and 93.8-100% , respectively . And it was higher than the homology between GXA and typical European strain LV , which was only 64.7% and 66.3%, respectively . The homology of nucleotide sequences and deduced amino acid sequences of M gene among GX
A and North American strains was 94.5-100% and 96.6-100% , respectively . It was higher than the homology between GXA and LV , which was only 69.2% and 78.9%, respectively. The homology of nucleotide sequences and deduced amino acid sequences of N gene among GXA and North American strains was 93.8-100% and 95.2-100% , respectively . It was higher than the homology between GXA and LV , which was only 66.3% and 63.9%, respectively. Furthermore , it was found that the homology was very high among GXA and VR-2332 , MLV , BJ-4 strains, but the homology was very low among GXA and LV .And the phylogenetic trees revealed that GXA strain perhaps has a close relation to VR-2332 , MLV and BJ-4 strains. These results suggested that GXA strain belongs to North American type , and probably comes from the vaccine strain.
E , M and N gene of GXA strain were obtained from recombinant plasmids pMD-E , pMD-M and pMD-N , then subcloned to prokaryotic expression vector PET-32a . And constructed the prokaryotic expression recombinant plasmids PET- E, PET-M and PET-N .They were identified by being digested with restriction endonucleases , the results showed that these recombinant plasmids were succeessfully constructed.
参考VR-2332和OH-1a株基因序列，设计了3对引物E1／E2、M1／M2、N1／N2，分别扩增GXA株的E、M、N基因。并进行克隆和测序。结果分别扩增出大小为668bp、582bp和443bp的目的片段。应用DNAstar生物学软件对所测得的序列进行处理后，得到GXA株的E、M和N基因ORF全长分别为603nt、524nt和372nt，分别编码200、174和123个氨基酸。将此3个基因序列分别与国内外已发表的ATCC VR-2332、MLV、BJ-4和LV等13个毒株相应基因序列进行了同源性比较。结果发现GXA株与美洲型毒株E基因的核苷酸及其推定的氨基酸的同源性分别为88.7-99.7％和93.8-100％，与欧洲型代表株—LV株的核苷酸及其推定的氨基酸的同源性分别为64.7％和66.3％；GXA株与美洲型毒株M基因的核苷酸及其推定的氨基酸的同源性分别为94.5-100％和96.6-100％，与LV株的核苷酸及其推定的氨基酸的同源性分别为69.2％和78.9％；GXA株与美洲型毒株N基因的核
广西大学2004届硕士毕业论文
昔酸及其推定的氨基酸的同源性分别为93.8一1 00%和95.2一1 00%，与LV株的核
昔酸及其推定的氨基酸的同源性分别为66.3%和63.9%。同源性分析显示，GxA
株与VR一2332、劫LV和BJ一4的同源性非常高，而GXA与LV同源性非常低。构建
的系统发育树分析，GXA与VR一2332、MLV及BJ一4株亲缘关系比较密切。从而表
明GXA株属于美洲型毒株，可能来源于疫苗株。
将GXA株的E、M、N基因从重组质粒 pMD一E、pMD一M、pMD一N中亚克隆至原
核表达载体PE丁一32a上，构建了原核表达重组质粒PE丁一E、pE丁一M和PE丁喇，并
通过酶切鉴定，证实构建成功。
In recent, some pig farms in Guangxi occur frequently an infectious disease , which characters are abortion , death of embryo , mummified fetuses , and respiratory problems in piglet . Pathogen of the disease was considered as porcine reproductive and respiratory syndrome virus (PRRSV) . In this study , we identified that the sample collected from the farm in Nanning was positive by RT-PCR with a pair of specific N gene primers of PRRSV . Based on it, permissive cells , Marc-145 cells were inoculated with the sample . After six blind passages , typical cytopathogenic effect (CPE) was observed on cells as well as the positive control .And PRRSV was positive in the cells and culture medium by RT-PCR . The first PRRSV strain in Guangxi was isolated successfully, named GXA strain . And its infection titer was 105.33 TCID50/ml.
According to VR-2332 and CH-1a strain , designed three pairs of primers E1/E2 , M1/M2 , N1/N2 ,which amplified the fragments of GXA strain's E , M , N gene , respectively . The amplified fragments were cloned and sequenced . The length of E , M and N gene fragment is 668 ,582 and 443 base pairs (bps), respectively . Processed the sequences by DNAstar software , obtained the length of E , M and N gene sequence is 603 ,524 and 372 nucleotides(nts), encoding 200 , 174 and 123 amino acids .respectively . Comparison of the homology among GXA , VR-2332 , MLV , BJ-4 , Lelystad virus (LV) and other 9 strains of PRRSV , the results shown that the homology of nucleotide sequences and deduced amino acid sequences of E gene among GXA and Norther American strains was 88.7-99.7% and 93.8-100% , respectively . And it was higher than the homology between GXA and typical European strain LV , which was only 64.7% and 66.3%, respectively . The homology of nucleotide sequences and deduced amino acid sequences of M gene among GX
A and North American strains was 94.5-100% and 96.6-100% , respectively . It was higher than the homology between GXA and LV , which was only 69.2% and 78.9%, respectively. The homology of nucleotide sequences and deduced amino acid sequences of N gene among GXA and North American strains was 93.8-100% and 95.2-100% , respectively . It was higher than the homology between GXA and LV , which was only 66.3% and 63.9%, respectively. Furthermore , it was found that the homology was very high among GXA and VR-2332 , MLV , BJ-4 strains, but the homology was very low among GXA and LV .And the phylogenetic trees revealed that GXA strain perhaps has a close relation to VR-2332 , MLV and BJ-4 strains. These results suggested that GXA strain belongs to North American type , and probably comes from the vaccine strain.
E , M and N gene of GXA strain were obtained from recombinant plasmids pMD-E , pMD-M and pMD-N , then subcloned to prokaryotic expression vector PET-32a . And constructed the prokaryotic expression recombinant plasmids PET- E, PET-M and PET-N .They were identified by being digested with restriction endonucleases , the results showed that these recombinant plasmids were succeessfully constructed.
引文
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