水牛PTHLH基因克隆分析及其真核表达载体构建

为揭示甲状旁腺激素样激素（parathyroid hormone-like hormone，PTHLH）基因对水牛繁殖性能的影响，本研究对水牛PTHLH基因进行克隆，并对其核苷酸和氨基酸序列进行生物信息学分析。以牛PTHLH基因为种子序列（GenBank登录号：NM_001290949），应用CE Design软件设计引物序列，运用PCR扩增和测序技术获得水牛完整编码区序列，使用DNAMAN、ProtParam、SOPMA、PSORTⅡ Prediction等在线软件分析PTHLH蛋白的一级结构、二级结构、三级结构与理化性质，并进行同源性比对分析及系统进化树构建。结果显示，试验克隆了水牛PTHLH基因完整编码区序列，该序列长为534 bp，可编码177个氨基酸。水牛PTHLH基因编码区核苷酸序列与黄牛、猪、马、山羊、绵羊和骆驼的同源性分别为98.3%、90.4%、90.1%、98.1%、97.5%和89.2%，物种之间同源性较高，系统进化树分析结果与其亲缘关系远近一致，表明水牛PTHLH基因编码区在进化过程中比较保守。蛋白理化性质分析显示，水牛PTHLH蛋白分子式为C₈₉₅H₁₄₅₁N₂₇₁O₂₆₆S₂，分子质量为2 885 u，半衰期为30 h，理论等电点（pI）为10.00，水溶液在280 nm处的消光系数为23 950，肽链N端为蛋氨酸（Met），不稳定系数为60.04，属于碱性不稳定蛋白；脂肪系数为72.15，总平均亲水性为－0.928，该蛋白属于不可溶性蛋白，亚细胞定位于细胞核、细胞质和线粒体。结构域预测结果显示，水牛PTHLH蛋白包含有1个PTH区域，同时还包含有1个低复杂度区域。二级结构分析显示，水牛PTHLH蛋白包含83个α-螺旋（46.89%）、17个延伸链（9.60%）、10个β-转角（5.66%）和67个无规则卷曲（37.85%），与三级结构预测结果相一致。试验构建了PTHLH基因真核表达载体pcDNA3.1-PTHLH，并通过电泳和测序验证了载体的准确性。PTHLH基因的成功克隆及其真核表达载体的成功构建为今后研究水牛PTHLH基因的功能和遗传特性提供了材料。

Cloning and Construction of Eukaryotic Expression of PTHLH Gene in Buffalo

In order to reveal the effect of parathyroid hormone-like hormone (PTHLH) gene on the reproductive ability of buffalo,buffalo PTHLH gene was cloned,and its nucleotide and amino acid sequences were analyzed by bioinformatics.Primers were designed by CE Design according to the sequence of cattle PTHLH gene in GenBank (accession No.:NM_001290949).The coding region of buffalo PTHLH gene was cloned by PCR amplification and sequencing technology.The primary structure,secondary structure,tertiary structure and physicochemical properties of PTHLH protein,and homology were analyzed,and phylogenetic tree of PTHLH was constructed by softwares such as DNAMAN,ProtParam,SOPMA and PSORTⅡ Prediction,et al.The results showed that PTHLH gene in buffalo was cloned,including a 534 bp length coding region and 177 amino acids.Compared with cattle,pigs,horses,goats,sheep and camels,the nucleotide sequence of gene coding region homology of PTHLH gene in buffalo were 98.3%,90.4%,90.1%,98.1%,97.5% and 89.2%,respectively.Thus,there was a high homology among different species,and the phyletic evolution was the same as their genetic relationship.The research indicated that PTHLH gene was conservative in the course of evolution.The physicochemical properties analysis result showed that the molecular formula of PTHLH protein was C₈₉₅H₁₄₅₁N₂₇₁O₂₆₆S₂,the molecular weight was 2 885 u,the half-life was 30 h,the theoretical electrical points (pI) was 10.00,the extinction coefficient of water solution at 280 nm was 23 950,the peptide chain N-terminal was Met,the unstable coefficient was 60.04,thus PTHLH protein belong to the basic unstable protein.The fat coefficient of PTHLH protein was 72.15,and the total average hydrophilicity was -0.928,so it belonged to insoluble protein.The protein subpopulations were located in the nucleus,cytoplasm and mitochondria.The structural domain prediction results showed that buffalo PTHLH protein contained a PTH region and a low complexity region.The secondary structure analysis showed that buffalo PTHLH protein contained 83 alpha helix (46.89%),17 extended chain (9.60%),10 beta turn (5.66%) and 67 random coil (37.85%),and consistent with the tertiary structure prediction.In this study,the eukaryotic expression vector was constructed,and the accuracy of the carrier was verified by electrophoresis and sequencing.In summary,the successful cloning of PTHLH gene and the successful construction of its eukaryotic expression vector provide materials for the future study of the function and genetic characteristics of PTHLH gene in buffalo.