广西猪瘟流行毒株E_2基因的克隆与序列分析
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摘要
研究采用反转录—聚合酶链式反应(RT—PCR)技术对两株分别从柳州(GXLZ)和南宁(GXNN)分离的广西流行猪瘟病毒(classical swine fever virus，CSFV)进行E_2全基因的扩增、克隆和测序。扩增片段长度为1090bp，编码364个氨基酸残基。应用DNAStar序列分析软件对所测的两个毒株GXLZ、GXNN与从GenBank调出的已知序列的国内外16株毒株的相应片段进行了同源性比较分析。结果表明所测的GXLZ、GXNN与HCLV的核苷酸序列同源性分别为82.0％、82.6％，与石门毒株(Shimen)的核苷酸同源性分别为83.5％、84.1％，在氨基酸序列比较中，GXNN、GXLZ与HCLV的同源性分别为88.7％、87.6％，与石汀洙的同源性分别为90.1％、89.0％。但广西的这两株流行毒株与德国的Paderborn和意大利的Italy株的核苷酸和氨基酸的同源性都比较高。GXNN、GXLZ与Paderborn株的核苷酸同源性为95.5％、95.6％。它们的氨基酸同源性高达96.7％、97.5％；而GXLZ、GXNN与Italy株的核苷酸序列同源性分别为95.0％、95.1％，氨基酸同源性则高达96.2％、97.0％。这一同源性分析结果表明广西流行猪瘟病毒与猪瘟兔化疫苗毒和标准石门强毒之间存在一定的差异。但可以推测广西近期流行猪瘟病毒与德国和意大利发生的猪瘟有密切的关系，有必要进行深入的调查研究。
GXLZ与GXNN和HCLV、Shimen的氨基酸序列对比发现，在E_2基因B、C高变的抗原区，GXLZ、GXNN在710、713、725、
孙石静：广西猪瘟流行毒株E。基因的克隆与序列分2
729、734和738位氨基酸均发生了变异。这些变异极有可能使广西
流行野毒逃避HCLV的免疫保护。
GXNN、GXLZ的E。基因与已知序列的16株国内外毒株的E。
基因相比较，建立了遗传进化树，可将CSFV分为两大群和一另类，
这与国内外学者做的结果相符。GxNN、GxLZ属匝群，与italy株
和Paderborn株以及Tkielll096株、39株同属11群。G皿Z、GXNN
、与属1群的中国主要参考毒株HCLV、Shimen在遗传关系上较远，
与Italy株和Paderborn株比较接近。在遗传进化关系上两野毒株与
疫苗毒株和标准强毒株相距较远，表明广西猪瘟流行毒株向远离疫
苗毒株的方向演变。
In this study, the whole E2 genes of two strains of Classical Swine Fever Virus (CSFV) isolated from Guangxi (GX) were amplified by reverse transcriptase polymerse chain reaction (RT-PCR) method and seqenced .The E2 gene fragments of CSFV were 1090 base pair in length and encoded 364 amino acid residues. Comparing the two epidemic strains (GXNN, GXLZ) with other 16 reference strains sequence reported in GenBank by software DNAStar, it was found that strains GXNN, GXLZ and Hog Cholera Lapinised Virus (HCLV) were 82.0%, 82.6% for nucleotide sequence and 88.7%, 87.6% for amino acid sequence in homology. The nucleotide homology among strains GXNN, GXLZ with Shimen were 83.5% and 84.1%; and that of amino acid were 90.1% and 89.0%. But compared the E2 genes of strains GXNN, GXLZ with Paderborn strain, the homology of nucleotide were 95.5% and 95.6%; and that of amino acid were 96.7%, 97.5% respectively. The homology between the two epidemic strains and Italy strain were higher and were 95.0%, 95.1% for nucleotide sequence and 96.3%, 97.0% for amino acid sequence. The result showed that there
were some differences among GX epidemic strains of CSFV, strains HCLV and Shimen. GX epidemic strains perhaps have a close relation to the CSFV occurred in Germany and Italy.
It also indicated that there were some mutations in 710,713,725, 729, 734 and 738 amino acid of B, C high variable region of strains GXNN, GXLZ by comparing the amino acid sequence. It was very impossible that those mutations of GX epidemic strains of CSFV can cause the incompleted protection of HCLV vaccine.
Computer- base phylogenetic tree among the two strains of GX and other 16 strains mentioned above were obtained by analyzing nucleotide sequences of E2 gene. It showed that these eighteen strains can be classified into two major groups and a special individual, which agreed with domestic and foreign scholors' conclusion. Strains GXNN, GXLZ belonged to group II ,which also included strains Italy, Paderborn, Thielll096 and 39. And another nine strains and strains HCLV, Shimen of two major reference strains of china belonged to group I. In addition, strain p97 was the special individual. The phylogenetic tree revealed that GX epidemic strains in recent years were genetically divergent from strains Shimen and HCLV.
GXLZ与GXNN和HCLV、Shimen的氨基酸序列对比发现，在E_2基因B、C高变的抗原区，GXLZ、GXNN在710、713、725、
孙石静：广西猪瘟流行毒株E。基因的克隆与序列分2
729、734和738位氨基酸均发生了变异。这些变异极有可能使广西
流行野毒逃避HCLV的免疫保护。
GXNN、GXLZ的E。基因与已知序列的16株国内外毒株的E。
基因相比较，建立了遗传进化树，可将CSFV分为两大群和一另类，
这与国内外学者做的结果相符。GxNN、GxLZ属匝群，与italy株
和Paderborn株以及Tkielll096株、39株同属11群。G皿Z、GXNN
、与属1群的中国主要参考毒株HCLV、Shimen在遗传关系上较远，
与Italy株和Paderborn株比较接近。在遗传进化关系上两野毒株与
疫苗毒株和标准强毒株相距较远，表明广西猪瘟流行毒株向远离疫
苗毒株的方向演变。
In this study, the whole E2 genes of two strains of Classical Swine Fever Virus (CSFV) isolated from Guangxi (GX) were amplified by reverse transcriptase polymerse chain reaction (RT-PCR) method and seqenced .The E2 gene fragments of CSFV were 1090 base pair in length and encoded 364 amino acid residues. Comparing the two epidemic strains (GXNN, GXLZ) with other 16 reference strains sequence reported in GenBank by software DNAStar, it was found that strains GXNN, GXLZ and Hog Cholera Lapinised Virus (HCLV) were 82.0%, 82.6% for nucleotide sequence and 88.7%, 87.6% for amino acid sequence in homology. The nucleotide homology among strains GXNN, GXLZ with Shimen were 83.5% and 84.1%; and that of amino acid were 90.1% and 89.0%. But compared the E2 genes of strains GXNN, GXLZ with Paderborn strain, the homology of nucleotide were 95.5% and 95.6%; and that of amino acid were 96.7%, 97.5% respectively. The homology between the two epidemic strains and Italy strain were higher and were 95.0%, 95.1% for nucleotide sequence and 96.3%, 97.0% for amino acid sequence. The result showed that there
were some differences among GX epidemic strains of CSFV, strains HCLV and Shimen. GX epidemic strains perhaps have a close relation to the CSFV occurred in Germany and Italy.
It also indicated that there were some mutations in 710,713,725, 729, 734 and 738 amino acid of B, C high variable region of strains GXNN, GXLZ by comparing the amino acid sequence. It was very impossible that those mutations of GX epidemic strains of CSFV can cause the incompleted protection of HCLV vaccine.
Computer- base phylogenetic tree among the two strains of GX and other 16 strains mentioned above were obtained by analyzing nucleotide sequences of E2 gene. It showed that these eighteen strains can be classified into two major groups and a special individual, which agreed with domestic and foreign scholors' conclusion. Strains GXNN, GXLZ belonged to group II ,which also included strains Italy, Paderborn, Thielll096 and 39. And another nine strains and strains HCLV, Shimen of two major reference strains of china belonged to group I. In addition, strain p97 was the special individual. The phylogenetic tree revealed that GX epidemic strains in recent years were genetically divergent from strains Shimen and HCLV.
引文
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