新疆双峰驼C3d基因cDNA的克隆与序列分析

Cloning and Sequence Analysis of cDNA of C3d Gene of Bactrian Camel from Xinjiang

【目的】克隆骆驼补体C3d基因,分析序列特征,为其佐剂效应研究提供依据。【方法】利用Trizol试剂,从骆驼肝脏组织提取总RNA,并反转录获得cDNA。设计C3d特异引物,应用PCR技术扩增C3d序列,构建到pMD18-T载体。将重组质粒转入感受态菌株并双酶切鉴定。使用ClustW,DNAstar,Swiss-Model软件对构建的重组C3d基因序列进行同源性比对,建立系统进化树并对其二级和三级结构进行预测和分析。【结果】采用骆驼C3d特异引物,PCR扩增获得大小为909 bp的骆驼补体C3d基因。构建至T载体并转入大肠杆菌,获得阳性转化克隆。克隆的新疆双峰驼C3d序列与骆驼科C3d基因之间的同源性在97%以上,与牛和猪C3d基因同源性为88%,与兔和人的C3d基因同源性为83%。对骆驼C3d蛋白采用Swiss-Model软件进行序列分析,预测该蛋白质的二级结构主要由α-螺旋和β-折叠组成,该蛋白可能具有良好的免疫原性和免疫结合活性。【结论】C3d基因在进化中较为保守,在骆驼免疫中可以采用亲缘关系较近的其他动物来源的C3d作为分子佐剂从而增强骆驼免疫效果。

【Objective】The C3d gene of camel complement was cloned and its sequence characteristics were analyzed to provide a basis for the study of its adjuvant effect.【Methods】Total RNA was extracted from camel liver using Trizol reagent,and cDNA was obtained by reverse transcription.C3d pecific primers were designed to amplify the C3d sequence by PCR technology,and the vector pMD18-T was constructed.The recombinant plasmid was transferred into competent strain and identified by double enzyme digestion.Clustw,DNA Star and Swiss-model software were used to compare the homology of the constructed recombinant C3d gene sequences,establish the phylogenetic tree,and predict and analyze its secondary and tertiary structures.【Results】Specific primers were designed and the 909 bp camel complement C3d gene was obtained by PCR amplification.T vector was constructed and transferred into Escherichia coli and a positive clone was obtained.Clustw was used to compare the DNA sequencing results with the sequences of bovine,pig,rabbit and human.The results showed that the cloned Bactrian camel C3d sequence had more than 97%homology with the C3d gene of Camelidae,and the homology with the C3d gene of bovine,pig,rabbit and human was gradually remote(88%,88%,83%and 83%,respectively).The sequence of C3d protein was analyzed by Swiss-model software,and the secondary structure of C3d protein was mainly composed ofα-helix andβ-fold,and it had good immunogenicity and protein-binding activity.【Conclusion】The C3d gene of camel complement was cloned.Sequence analysis showed that the C3d gene was relatively conserved in evolution,which provided the experimental basis and technical reference for the adjuvant effect of the gene and the subsequent research.