蓝舌病新疆分离株VP7蛋白编码基因序列分析及一步法RT-PCR方法的建立

doi:10.6048/j.issn.1001-4330.2024.01.027

摘要/Abstract

摘要：

【目的】分析蓝舌病新疆分离株编码VP7蛋白S7基因保守序列,建立蓝舌病新疆分离株一步法RT-PCR方法,为新疆BTV分子流行病学调查及防控提供技术支持。【方法】采用二代测序技术获得新疆分离株S7基因序列,登陆GenBank对序列进行同源性Blast比对,用软件MEGA 5.0分析序列差异,根据蓝舌病新疆分离株编码VP7蛋白S7基因保守序列,运用软件Oligo6.0设计引物,对蓝舌病新疆分离株S7基因进行RT-PCR扩增,并验证该方法的特异性及敏感性。【结果】蓝舌病中国新疆分离株编码VP7蛋白S7基因序列与哈尔滨报道BTV分离株S7基因片段相似度较高为89.64%,与中国云南报道BTV-29型分离株、蒙古国BTV分离株S7基因片段相似度分别为87.49%和87.39%;与德国BTV分离株S7基因片段相似度为80.70%,其余均无同源性序列。中国新疆分离株S7基因片段序列与4条同源序列间存在26处核苷酸差异,9处氨基酸差异。【结论】建立的蓝舌病中国新疆分离株S7基因片段一步法RT-PCR方法具有良好的特异性,仅BTV中国新疆分离株扩增获得目的条带,该方法的敏感性为 4.0×10³copies/mL。

关键词: 蓝舌病; VP7蛋白; RT-PCR

Abstract:

【Objective】 According to conserved sequence of VP7 protein coding S7 gene of bluetongue isolated in Xinjiang,a one-step RT-PCR method was established,the results provides technical support for molecular epidemiological investigation and prevention of BTV in Xinjiang. 【Methods】 The S7 gene sequence which was obtained by second-generation sequencing,were compared the homology by Blast in GenBank,and analyzed the difference by Mega 5.0. Primers were designed according to conserved sequence of S7 gene encoding VP7 protein of bluetongue isolated in Xinjiang by Oligo 6.0. One-step RT-PCR method was established,then specificity and sensitivity was verified. 【Results】 Sequence comparison results showed that S7 gene of bluetongue isolated in Xinjiang has highest homology with BTV isolated fragment reported by Harbin,it was 89.64%.The sequence similarity between S7 gene of bluetongue isolated in Xinjiang and other fragment including BTV-29 reported by Yunnan,BTV isolated from Mongolia and German were87.49%,87.39% and 80.70% respectively.Excpt those squences,there was no more homologous sequences.The results showed that there were 26 nucleotide variation and 9 amino acid variation between the S7 gene fragment sequence of Xinjiang isolate and 4 homologous sequences. 【Conclusion】 One-step RT-PCR was able to specifically detect S7 gene of bluetongue isolated in Xinjiang,simultaneously with the detection limit of 4.0×10³copies/mL. But no genomic RNA or DNA were amplified from BVDV and IBRV.

Key words: bluetongue; VP7 protein; RT-PCR

中图分类号:

S188

马晓菁, 谷文喜, 叶锋, 刘帅, 刘丽娅, 谢彩云, 钟旗, 易新萍. 蓝舌病新疆分离株VP7蛋白编码基因序列分析及一步法RT-PCR方法的建立[J]. 新疆农业科学, 2024, 61(1): 253-259.

MA Xiaojing, GU Wenxi, YE Feng, LIU Shuai, LIU Liya, XIE Caiyun, ZHONG Qi, YI Xinping. Sequence analysis and one-step RT-PCR establishment of VP7 protein coding gene of bluetongue isolated in Xinjiang[J]. Xinjiang Agricultural Sciences, 2024, 61(1): 253-259.

图/表 4

表1 一步法RT-PCR扩增引物

Tab.1 One step RT-PCR amplification of primers

引物名称 Primers	目的基因 Target gene	序列(5’-3’) Sequence(5’-3’)	片段大小(bp) Fragment size(bp)
F-7X	BTV S7	CAGCAAGGGCGCTCACTGTGATG	1010
F-7R	BTV S7	GCGTGTGAGCGGGCCCGGCATAGCATTCACA	1010

图1 BTV新疆分离株巢式PCR扩增结果注:M:DNAMarkerDM2000;1-2:BHK 细胞提取RNA;3-4:BTV新疆分离株提取RNA

Fig.1 The results of nested PCR amplification of BTV Xinjiang isolates Note:M:DNAMarkerDM2000;1-2:RNA extraction from BHK cells;3-4:RNA extraction from BTV Xinjiang isolate

图2 BTV新疆分离株一步法RT-PCR扩增注:M:DNAMarkerDL2000; 1:BHK细胞提取RNA;2:ddH20空白对照; 3:牛病毒性腹泻病毒提取RNA;4:牛传染性鼻气管炎病毒提取DNA;5:BTV新疆分离株提取RNA

Fig.2 One-step RT-PCR amplification results of BTV Xinjiang isolate Note:M:DNAMarkerDL2000; 1:RNA extraction from BHK cells;2:ddH20 blank control; 3:RNAextraction from Bovine Viral Diarrhoes Mucosal Virus;4:DNA extraction from Infectious Bovine Rhinotracheitis Virus;5:RNA extraction from BTV Xinjiang isolate

图3 BTV新疆分离株一步法RT-PCR敏感性注:M:DNAMarkerDL2000;1:BHK细胞提取RNA;2:标准质粒4×106copies/μL;3:标准质粒4×105copies/μL;4:标准质粒4×104copies/μL;5:标准质粒4×103copies/μL;6:标准质粒4×102copies/μL

Fig.3 Sensitivity results of one-step RT-PCR for BTV Xinjiang isolate Note:M:DNAMarkerDL2000;1:RNA extraction from BHK cells;2:Standard plasmids 4×106copies/μL;3:Standard plasmids 4×105copies/μL;4:Standard plasmids 4×104copies/μL;5:Standard plasmids 4×103copies/μL;6:Standard plasmids 4×102copies/μL

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