刚地弓形虫P30基因的克隆及编码蛋白结构与抗原表位的生物信息学分析

Cloning of P30 gene from Toxoplasma gondii and analyzing of encoded protein structure and antigen epitope from bioinformatics

目的克隆刚地弓形虫P30基因,从生物信息学角度分析P30基因编码蛋白的结构与抗原表位,预测该蛋白的免疫原性。方法提取刚地弓形虫(Toxoplasma gondii)RH株速殖子总RNA,进行RT-PCR扩增,扩增产物经NheI和AflⅡ双酶切后与真核表达载体PVAXI连接,转化后通过菌落PCR、双酶切和测序鉴定。使用Protparam、Protscal、SOSU、SWISS-MODEL、SOPMA、Bcepred和TMHMM等在线分析工具,分析P30蛋白的物理和化学性质、亲水性、疏水性、二级结构、表面可及性、柔韧性、细胞定位、跨膜结构域、信号肽、翻译后修饰位点、结构域、功能域、抗原表位和三级结构。结果RT-PCR扩增大小为789 bp,菌落PCR显示,在约789 bp处出现特异性扩增片段,与预期大小相符,阳性PVAXI-P30重组质粒经Nhel和AflⅡ双酶切获得大小为789 bp和3000 bp的两条条带,大小与目的基因和载体片段相等,测序结果显示,P30基因大小为789 bp,与GenBank的P30基因(登录号为X14080.1)核苷酸序列一致性为100%。P30蛋白含有336个氨基酸,分子式为C_(1525)H_(2459)N_(409)O_(477)S_(21),相对分子质量为34.83×10^(3),理论等电点(8.34),不稳定指数(36.70),脂溶性指数(80.15),消光系数(20970),A_(280)(0.602),P30亲水性氨基酸分布在整条链上,具有两亲性,在P30蛋白的336个氨基酸中,α-螺旋(Hh)占22.32%,β-折叠(Ee)占26.49%,β-转角(Tt)占8.04%,无规则卷曲(Cc)占43.15%,表面可及性参数得分≥1.9的区域是11个,柔韧性参数得分≥2.0的区域和亲水性参数得分≥1.9的区域均有6个,翻译后修饰位点有6个,保守结构域有5类,潜在B细胞抗原表位共13个,8个潜在限制性CTL细胞抗原表位,13个潜在辅助T细胞抗原表位,P30蛋白最有可能是定位于真核细胞线粒体内的可溶性表达蛋白。结论刚地弓形虫P30基因编码的蛋白为可溶性表达蛋白,具有免疫原性,可为弓形虫病疫苗的研制提供理论基础。

Objective Cloning the Toxoplasma gondii P30 gene and analyzing the structure and epitope of Toxoplasma gondii P30 gene encoding protein from the perspective of bioinformatics,to predict the immunogenicity of the protein.Methods Total RNA was extracted from tachyzoites of RH strain of Toxoplasma gondii,primers were designed according to the open reading frame of the P30 gene(Accession No.X14080.1),RT-PCR amplification,the amplified product of RT-PCR was digested with double restriction enzyme Nhel and AflⅡ,and ligated into the eukaryotic expression vector PVAXI.The recombinant plasmid was transferred into E.coli XL-Blue,the positive clones was selected through the colony-PCR and confirmed by the double restriction enzyme digestion and sequencing.Using the online biological software as Protparam,Protscal,SOSU,SWISS-MODEL,SOPMA,Bcepred and TMHMM,the physical and chemical properties(including molecular weight,isoelectric point,stability index,amino acid composition,extinction coefficient,etc.),hydrophilicity,hydrophobicity,secondary structure,surface accessibility,flexibility,cell location,transmembrane domain,signal peptide,post-translational modification sites,structural domain,functional domain,Epitopes(B cell epitopes,T cell epitopes)and tertiary structure of the P30 protein were analyzed.Results The RT-PCR products of P30 were 789 bp consistent with expected size,by agarose gel electrophoresis,the colony PCR products appear at the place of 789 bp in line with the expected size,the positive PVAXI-P30 recombinant plasmid were double digested to obtain two bands with sizes of 789 bp and 3000 bp,which were equal to the target gene and vector fragment,sequencing results showed that obtained P30 was 789 bp,compared with the existing sequence of Toxoplasma gondii P30 gene on GenBank,sequence consistency of 100%.P30 protein had 336 amino acids,the molecular formula was C_(1525)H_(2459)N_(409)O_(477)S_(21),the molecular mass was 34.83×10^(3),the theoretical isoelectric point(8.34),the instability index(36.70),the fat solubility index(80.15),the extinction coefficient(20970),A_(280)(0.602),the P30 protein hydrophilic amino acids were distributed in the entire chain and were amphiphilic,the average hydrophobicity coefficient of the P30 protein was 0.129,among the 336 amino acids of the P30 protein,α-helix(Hh)accounts for 22.32%,β-sheet(Ee)accounts for 26.49%,β-turn(Tt)accounts for 8.04%,and random coils(Cc)accounts for 43.15%,the surface area of≥1.9 parameter score were eleven,the regions with flexibility parameter score≥2.0 were six,the regions with hydrophilicity parameter score≥1.9 were six,post-translational modification sites were six,conserved domain class were five,potential B cell epitopes were thirteen,potential restricted CTL cell epitopes were eight,potential helper T cell epitopes were thirteen,the P30 protein had the transmembrane region and the signal peptide sequencethe,and was most likely a soluble protein expressed in the mitochondria of eukaryotic cells.Conclusion The protein encoded by the P30 gene of Toxoplasma gondii is a soluble protein expressed and has immunogenicity,which lays the foundation for subsequent research on the P30 protein.