新疆双峰驼C3d基因cDNA的克隆与序列分析

doi:10.6048/j.issn.1001-4330.2023.02.029

新疆农业科学 ›› 2023, Vol. 60 ›› Issue (2): 493-500.DOI: 10.6048/j.issn.1001-4330.2023.02.029

• 林业·草业·畜牧兽医 • 上一篇下一篇

新疆双峰驼C3d基因cDNA的克隆与序列分析

娜斯拜·阿卜杜瓦哈普(), 高晓娟, 坤杜孜阿依·阿布都沙拉木, 李江伟()

新疆大学生命科学与技术学院/省部共建新疆生物资源基因工程重点实验室,乌鲁木齐 830046

收稿日期:2022-06-23 出版日期:2023-02-20 发布日期:2023-03-31
通信作者: 李江伟(1967-),男,新疆乌鲁木齐人,教授,硕士生导师,研究方向为抗体工程及肿瘤免疫治疗,(E-mail)jwli67@sina.com
作者简介:娜斯拜·阿卜杜瓦哈普(1994-),女,新疆奇台人,硕士研究生,研究方向为生化与分子生物学,(E-mail)1535313706@qq.com
基金资助:
国家自然科学基金“基于高通量测序对骆驼抗体库的多样性分析和数据挖掘及新纳米抗体的发现”(31570935)

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

Nasibai Abuduwahapu(), GAO Xiaojuan, Kunduziayi Abudushalamu, LI Jiangwei()

Xinjiang Key Laboratory of Biological Resources and Genetic Engineering / College of Life Science and Technology, Xinjiang University, Urumqi 830046, China

Received:2022-06-23 Online:2023-02-20 Published:2023-03-31
Correspondence author: LI Jiangwei (1967-), male, Xuchang, Henan province. Ph.D., researcher, master superivisor,(E-mail)jwli67@sina.com
Supported by:
General Project of National Natural Science Foundation of China" Diversity Analysis and Data Mining of Camel Antibody Library Based on High-Throughput Sequencing and Discovery of New Nano Antibodies"(31570935)

摘要/Abstract

摘要：

【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。

关键词: 骆驼C3d; PCR; 基因克隆; 序列分析; 结构预测

Abstract:

【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 of α-helix and β-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.

Key words: camel C3d; PCR; gene cloning; sequence analysis; structure prediction

中图分类号:

S852.4

娜斯拜·阿卜杜瓦哈普, 高晓娟, 坤杜孜阿依·阿布都沙拉木, 李江伟. 新疆双峰驼C3d基因cDNA的克隆与序列分析[J]. 新疆农业科学, 2023, 60(2): 493-500.

Nasibai Abuduwahapu, GAO Xiaojuan, Kunduziayi Abudushalamu, LI Jiangwei. Cloning and Sequence Analysis of cDNA of C3d Gene of Bactrian Camel from Xinjiang[J]. Xinjiang Agricultural Sciences, 2023, 60(2): 493-500.

图/表 10

参考文献 18

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引物 Primer	序列(5'-3') Sequence(5'-3')	C3中位置 Location in the C3	产物长度 The length of product(bp)
C3d-primer-F	cgatccatggTAAAGCACCTCATCGTGACCCCGT	3 086	909
C3d-primer-R	attactcgagGCGGCTGGGCAGGTGGATGGAC	3 995	909

引物 Primer	序列(5'-3') Sequence(5'-3')	C3中位置 Location in the C3	产物长度 The length of product(bp)
C3d-primer-F	cgatccatggTAAAGCACCTCATCGTGACCCCGT	3 086	909
C3d-primer-R	attactcgagGCGGCTGGGCAGGTGGATGGAC	3 995	909

结构分析 Structure analysis	预测结果 Predicted results
α-螺旋 α-Screw	12-32 37-44 48-54 110-114 122-140 157-162 176-204 217-227 254-268 273-280
β-折叠 β-fold	1-12 59-93 106-109 163-197 172-175 232-244 276-282
T-转角 T-corner	33-36 44-47 55-58 96-99 102-105 118-121 141-144 146-149 153-156 168-171 205-208 228-231 246-253 269-272 283-286
卷曲区 Crimp zone	1-2 43-45 55-56 58 96-97 99 102 105-106 115-118 145-148 151-153 155-156 168 195 200-201 205 209-210 231-235 240 249-253 268-269 283-284
亲水区 Hydrophilic zone	11-32 33-49 58-60 92-104 105-109 113-126 131-144 145-155 157-160 161-162 163-170 171-172 180-181 183-186 187-189 194-216 230-232 234-235 237-254 263-277 282-283 284-287
柔韧性 Flexibility	14-18 23-27 33-39 43-45 56-59 79-81 93-99 102-106 115-123 136-139 142-157 167-169 183-186 195-210 215-216 227-234 240-253 266-274
抗原性 Antigenicity	12-19 12-37 41-48 55-60 91-107 113-126 135-160 164-172 181-188 189-190 197-213 215-217 227-235 239-254 265-278 283-287
表面可及性 Surface accessibility	11-19 23-28 34-39 43-47 93-98 117-125 165-170 197-212 239-245 264-274

结构分析 Structure analysis	预测结果 Predicted results
α-螺旋 α-Screw	12-32 37-44 48-54 110-114 122-140 157-162 176-204 217-227 254-268 273-280
β-折叠 β-fold	1-12 59-93 106-109 163-197 172-175 232-244 276-282
T-转角 T-corner	33-36 44-47 55-58 96-99 102-105 118-121 141-144 146-149 153-156 168-171 205-208 228-231 246-253 269-272 283-286
卷曲区 Crimp zone	1-2 43-45 55-56 58 96-97 99 102 105-106 115-118 145-148 151-153 155-156 168 195 200-201 205 209-210 231-235 240 249-253 268-269 283-284
亲水区 Hydrophilic zone	11-32 33-49 58-60 92-104 105-109 113-126 131-144 145-155 157-160 161-162 163-170 171-172 180-181 183-186 187-189 194-216 230-232 234-235 237-254 263-277 282-283 284-287
柔韧性 Flexibility	14-18 23-27 33-39 43-45 56-59 79-81 93-99 102-106 115-123 136-139 142-157 167-169 183-186 195-210 215-216 227-234 240-253 266-274
抗原性 Antigenicity	12-19 12-37 41-48 55-60 91-107 113-126 135-160 164-172 181-188 189-190 197-213 215-217 227-235 239-254 265-278 283-287
表面可及性 Surface accessibility	11-19 23-28 34-39 43-47 93-98 117-125 165-170 197-212 239-245 264-274

物种 Species	分子量 Molecular weight	等电点 Isoelectric point	pH7时的电荷 The charge at pH7	疏水性平均值 Mean hydrophobicity	抗原性指数 Antigenicity index
新疆双峰驼Xinjiang Bactrian camel	32 185.02	6.4	-3.13	0.13	0.53
双峰驼Bactrian camel	33 951.1	6.25	-2.99	0.12	0.43
单峰驼Dromedary camel	33 951.1	6.25	-2.99	0.12	0.43
羊驼Alpaca	33 865.05	6.7	-0.99	0.1	0.39
野驼Wild camel	33 670.65	6.03	-3.99	0.07	0.44
人People	33 915	6.37	-1.49	0.16	0.39
鼠Rat	34 015.9	5.49	-6.15	0.18	0.4
猪Pig	34 160.23	6.13	-3.15	0.19	0.47
家兔Rabbit	34 255.28	7.85	1.84	0.2	0.37
牛Cattle	34 457.36	7.38	0.84	0.36	0.5

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

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