猪瘟兔化弱毒疫苗株细胞毒E2基因的克隆与序列分析
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摘要
本论文对猪瘟兔化弱毒疫苗株在猪睾丸(ST)细胞和牛睾丸(BT)细胞上培养适应性、病毒增殖能力及细胞的纯净性方面进行了系统研究;从分子水平的角度,分析和比较了猪瘟兔化弱毒疫苗株ST细胞和BT细胞毒的主要保护性抗原E2蛋白在抗原编码区内氨基酸的变异情况,同时对其核苷酸序列和推导的氨基酸序列进行同源性分析,并应用遗传进化树理论对其E2基因的来源及其分子遗传衍化规律进行研究。
本实验所用的猪瘟兔化弱毒疫苗株分别在ST细胞和BT细胞上培养增殖后,从病毒感染的细胞液中直接提取核酸。应用Oligo(6.0版本)引物设计分析软件设计猪瘟病毒(CSFV)E2基因片段的特异性PCR引物,通过反转录-聚合酶链式反应(RT-PCR)方法扩增两种猪瘟兔化弱毒疫苗株细胞(ST、BT)毒E2基因的cDNA,PCR产物经分离纯化后克隆到pMD 18-T载体中,重组质粒经PCR鉴定和酶切鉴定纯化后进行序列测定,然后应用DNAstar软件进行序列的拼接、序列信息编辑和分析。同时从GenBank中下载了猪瘟兔化弱毒疫苗株PK15细胞毒株、石门强毒标准株以及猪瘟标准C-株的E2基因序列,分析了猪瘟兔化弱毒疫苗株ST、BT细胞毒E2基因的核苷酸序列及推导的氨基酸序列的同源性和差异性,绘制遗传进化树,以阐明猪瘟兔化弱毒疫苗株在适应了ST细胞和BT细胞后,ST、BT细胞毒主要保护性抗原的变异情况及其遗传演化规律。
研究结果发现,猪瘟兔化弱毒疫苗株经ST细胞和BT细胞培养增殖后,猪瘟兔化弱毒疫苗株不仅在BT细胞上具有良好的适应性,而且在ST传代细胞上生长状态良好,并保持了细胞的纯净性和良好的病毒增殖能力。
通过对猪瘟兔化弱毒疫苗株ST、BT细胞毒E2基因的核苷酸和氨基酸序列进行比较和分析,发现猪瘟兔化弱毒疫苗株BT细胞、ST细胞毒与标准参考毒株AY663656-C81(猪瘟兔化弱毒疫苗株PK15细胞毒)同源性相对较高,核苷酸和氨基酸序列同源性分别达到99.0%～99.4%和98.7%～99.7%,与标准参考毒株Z46258-C株的核苷酸和氨基酸序列的同源性次之,即分别为98.7%～98.9%和98.4%～99.0%;绘制其E2基因的遗传进化树,明确了两种猪瘟兔化弱毒疫苗株ST、BT细胞毒的E2基因与AY663656-C81和标准Z46258-C疫苗株同属于一个基因群,起源于标准Shimen猪瘟强毒株。
而且,在其E2蛋白抗原区的氨基酸差异分析比较中,发现猪瘟兔化弱毒疫苗株ST细胞毒与标准C疫苗株在诱导中和抗体的A、B、C三个抗原区内无一氨基酸差异,猪瘟兔化弱毒疫苗株BT细胞毒在诱导中和抗体的A、B、C、D四个抗原区内与标准C疫苗株的氨基酸仅有细微差异,证实了猪瘟病毒分子结构的遗传稳定性,进一步从分子生物学的角度显示了猪瘟兔化弱毒疫苗株在适应ST细胞、BT细胞后,并没有影响疫苗细胞毒的抗原性。
总之,本研究系统地阐明了猪瘟兔化弱毒疫苗株在猪睾丸细胞和牛睾丸细胞上培养增殖情况,从分子遗传演化分析的角度,进一步明确猪瘟兔化弱毒疫苗株ST、BT细胞毒的主要保护性抗原E2蛋白在A、B、C、D四个抗原区内的变异情况,确保疫苗种毒良好的细胞适应性和抗原性,为今后猪瘟疫苗的工艺优化和理想的免疫效果提供重要的理论依据。
In this study, Hog cholera Virus lapinized chinese strain can grow in Bovine Testicle Cell and Swine Testis Cell, and detect growth characteristics, HCLV multiplication and no others viruses. The further study of genetic relationship between the major protective antigen encoding regions of E2 genes of ST cell strain and BT cell strain was done by molecular biology heredity methods.
The nucleic acids of Hog cholera viruses conducted in the research were extracted from the viruses propagated in ST cell and BT cell. E2 Gene of different cell strains infected by HCLV were amplified by RT-PCR (Reverse transcription-polymerase chain reaction). The cDNAs were cloned into pMD18-T vector and identified. The sequencing products were purified and sequenced. Finally, using DNAStar software was used for data analysis, sequence translation, phylogenetic tree drawing and molecular evolution analysis.
The results indicated that HCLV can multiplicated in Bovine Testicle Cell and Swine Testis Cell,and cell’s stabilization growth characteristics, and no exogenous viruses.
Their nucleotide sequences were determined and the amino acid sequences were deduced. Compared with the corresponding region of AY663656-C81, their homologies among seven strains were very higher. That is, their nucleotide sequence homologies and amino acid homologies were 99.0%～99.4%,98.7%～99.7% respectively. And the nucleotide sequence homologies and amino acid homologies were 99.0%～99.4%, 98.7%～99.7% respectively, compared with Z46258-C. The phylogenetic trees revealed that E2 genes of ST cell strain and BT cell strain, AY663656-C81 and Z46258-C conducted in this study belonged to the same group, derived from AF092448-Shimen.
Furthermore, the variation analysis of amino-acid in the major protective antigen encoding regions of E2 genes of all strains, showed that E2 genes of ST cell strain be none mutation in A,B,C and D antigen regions, While there is subtle difference in E2 genes of BT cell strain’s A,B,C and D antigen regions. The research confirmed that E2 genes of ST cell strain and BT cell strain infected by HCLV were genetically stable.
In conclusion, this study elucidated HCLV multiplication be cultured in Bovine Testicle Cell and Swine Testis Cell,and E2 genes of ST cell and BT cell strain be none or subtle difference in A,B,C and D antigen regions by molecular heredity evolution methods. The research ensured vaccine virus in ST cell and BT cell favourable suitability and antigenicity. Of particular significance, these would provide the theory support for the possibility and development trend of the classical swine fever vaccine in ideal immunological effect.
本实验所用的猪瘟兔化弱毒疫苗株分别在ST细胞和BT细胞上培养增殖后,从病毒感染的细胞液中直接提取核酸。应用Oligo(6.0版本)引物设计分析软件设计猪瘟病毒(CSFV)E2基因片段的特异性PCR引物,通过反转录-聚合酶链式反应(RT-PCR)方法扩增两种猪瘟兔化弱毒疫苗株细胞(ST、BT)毒E2基因的cDNA,PCR产物经分离纯化后克隆到pMD 18-T载体中,重组质粒经PCR鉴定和酶切鉴定纯化后进行序列测定,然后应用DNAstar软件进行序列的拼接、序列信息编辑和分析。同时从GenBank中下载了猪瘟兔化弱毒疫苗株PK15细胞毒株、石门强毒标准株以及猪瘟标准C-株的E2基因序列,分析了猪瘟兔化弱毒疫苗株ST、BT细胞毒E2基因的核苷酸序列及推导的氨基酸序列的同源性和差异性,绘制遗传进化树,以阐明猪瘟兔化弱毒疫苗株在适应了ST细胞和BT细胞后,ST、BT细胞毒主要保护性抗原的变异情况及其遗传演化规律。
研究结果发现,猪瘟兔化弱毒疫苗株经ST细胞和BT细胞培养增殖后,猪瘟兔化弱毒疫苗株不仅在BT细胞上具有良好的适应性,而且在ST传代细胞上生长状态良好,并保持了细胞的纯净性和良好的病毒增殖能力。
通过对猪瘟兔化弱毒疫苗株ST、BT细胞毒E2基因的核苷酸和氨基酸序列进行比较和分析,发现猪瘟兔化弱毒疫苗株BT细胞、ST细胞毒与标准参考毒株AY663656-C81(猪瘟兔化弱毒疫苗株PK15细胞毒)同源性相对较高,核苷酸和氨基酸序列同源性分别达到99.0%～99.4%和98.7%～99.7%,与标准参考毒株Z46258-C株的核苷酸和氨基酸序列的同源性次之,即分别为98.7%～98.9%和98.4%～99.0%;绘制其E2基因的遗传进化树,明确了两种猪瘟兔化弱毒疫苗株ST、BT细胞毒的E2基因与AY663656-C81和标准Z46258-C疫苗株同属于一个基因群,起源于标准Shimen猪瘟强毒株。
而且,在其E2蛋白抗原区的氨基酸差异分析比较中,发现猪瘟兔化弱毒疫苗株ST细胞毒与标准C疫苗株在诱导中和抗体的A、B、C三个抗原区内无一氨基酸差异,猪瘟兔化弱毒疫苗株BT细胞毒在诱导中和抗体的A、B、C、D四个抗原区内与标准C疫苗株的氨基酸仅有细微差异,证实了猪瘟病毒分子结构的遗传稳定性,进一步从分子生物学的角度显示了猪瘟兔化弱毒疫苗株在适应ST细胞、BT细胞后,并没有影响疫苗细胞毒的抗原性。
总之,本研究系统地阐明了猪瘟兔化弱毒疫苗株在猪睾丸细胞和牛睾丸细胞上培养增殖情况,从分子遗传演化分析的角度,进一步明确猪瘟兔化弱毒疫苗株ST、BT细胞毒的主要保护性抗原E2蛋白在A、B、C、D四个抗原区内的变异情况,确保疫苗种毒良好的细胞适应性和抗原性,为今后猪瘟疫苗的工艺优化和理想的免疫效果提供重要的理论依据。
In this study, Hog cholera Virus lapinized chinese strain can grow in Bovine Testicle Cell and Swine Testis Cell, and detect growth characteristics, HCLV multiplication and no others viruses. The further study of genetic relationship between the major protective antigen encoding regions of E2 genes of ST cell strain and BT cell strain was done by molecular biology heredity methods.
The nucleic acids of Hog cholera viruses conducted in the research were extracted from the viruses propagated in ST cell and BT cell. E2 Gene of different cell strains infected by HCLV were amplified by RT-PCR (Reverse transcription-polymerase chain reaction). The cDNAs were cloned into pMD18-T vector and identified. The sequencing products were purified and sequenced. Finally, using DNAStar software was used for data analysis, sequence translation, phylogenetic tree drawing and molecular evolution analysis.
The results indicated that HCLV can multiplicated in Bovine Testicle Cell and Swine Testis Cell,and cell’s stabilization growth characteristics, and no exogenous viruses.
Their nucleotide sequences were determined and the amino acid sequences were deduced. Compared with the corresponding region of AY663656-C81, their homologies among seven strains were very higher. That is, their nucleotide sequence homologies and amino acid homologies were 99.0%～99.4%,98.7%～99.7% respectively. And the nucleotide sequence homologies and amino acid homologies were 99.0%～99.4%, 98.7%～99.7% respectively, compared with Z46258-C. The phylogenetic trees revealed that E2 genes of ST cell strain and BT cell strain, AY663656-C81 and Z46258-C conducted in this study belonged to the same group, derived from AF092448-Shimen.
Furthermore, the variation analysis of amino-acid in the major protective antigen encoding regions of E2 genes of all strains, showed that E2 genes of ST cell strain be none mutation in A,B,C and D antigen regions, While there is subtle difference in E2 genes of BT cell strain’s A,B,C and D antigen regions. The research confirmed that E2 genes of ST cell strain and BT cell strain infected by HCLV were genetically stable.
In conclusion, this study elucidated HCLV multiplication be cultured in Bovine Testicle Cell and Swine Testis Cell,and E2 genes of ST cell and BT cell strain be none or subtle difference in A,B,C and D antigen regions by molecular heredity evolution methods. The research ensured vaccine virus in ST cell and BT cell favourable suitability and antigenicity. Of particular significance, these would provide the theory support for the possibility and development trend of the classical swine fever vaccine in ideal immunological effect.
引文
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