K亚群禽白血病病毒分离株基因测序与鉴定分析  

Gene sequencing and identification analysis of the isolates of Avian leukemia virus subgroup K

作　　者：沈晓鹏[1] 曹斌[1] 王传峰[1] 陈则东[1] 马跃 朱建平[1] 王成丽[1] 叶建强[2] 俞燕 李拓凡 张俊 SHEN Xiaopeng;CAO Bin;WANG Chuanfeng;CHEN Zedong;MA Yue;ZHU Jianping;WANG Chengli;YE Jianqiang;YU Yan;LI Tuofan;ZHANG Jun(Jiangsu Agri-animal Husbandry Vocational College,Taizhou 225300,China;College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,China)

机构地区：[1]江苏农牧科技职业学院,江苏泰州225300 [2]扬州大学兽医学院,江苏扬州225009

出　　处：《黑龙江畜牧兽医》2021年第23期88-93,155,共7页Heilongjiang Animal Science And veterinary Medicine

摘　　要：为了对从江苏省某种鸡场保种的崇仁麻鸡血浆中分离的禽白血病病毒(ALV)进行序列测定和亚群鉴定,试验采用ELISA法检测血浆样品p27抗原,并用从阳性血浆中分离的病毒接种DF1细胞进行免疫荧光检测,提取前病毒DNA,用ALV亚群鉴定引物进行PCR鉴定,前病毒DNA分3段扩增并测序,对测序结果进行拼接,用MEGA 7.0软件以NJ法对分离株病毒gp85基因编码的氨基酸序列和参考株进行遗传进化分析,采用DNAStar 7.0软件以Clustal W法对参考株与分离株病毒的gp37基因进行比对分析。结果表明:有1份样品的p27抗原ELISA反应呈阳性,免疫荧光检测也呈阳性,将该分离株命名为JS20CR13。K亚群引物PCR检测出大小为560 bp的目的片段。前病毒全基因组总长7 481 bp,其中env基因大小为1 617 bp(gp85基因1 008 bp,编码336个氨基酸;gp37基因大小为609 bp,编码203个氨基酸)。与各参考株对比,分离株JS20CR13的gp85基因序列与K亚群参考株同源性为94.0%~99.0%,明显高于其他亚群;gp85基因编码氨基酸序列与已经鉴定为ALV-K的参考株病毒位于同一个进化分支,而与其他亚群的遗传进化距离较远。分离株JS20CR13的gp37基因序列与ALV-K参考株JS14CZ01同源性最高,达99.0%。说明分离株JS20CR13与已经鉴定为ALV-K的参考株病毒位于同一个进化分支,分离株为K亚群ALV。In order to carry out of sequence determination and subgroups dentifiation from the plasma Chongren Ma chicken protected by a certain chicken farm in Jiangsu Province, the p27 antigen of plasma samples was detected by ELISA, and the ALV was isolated from the positive plasma, which was inoculated in DF1 cells for IFA test. The proviral DNA was extracted, and PCR identification was performed with ALV subgroup identification primers;the proviral DNA was amplified and sequenced in three segments, and the sequencing results were spliced;the genetic evolution analysis of the amino acid sequence encoded by the gp85 gene of the isolated virus and the reference strain was carried out using the NJ method in the MEGA 7.0 software, and the DNAStar 7.0 software was used to compare and analyze the gp37 gene of the reference strain and the isolated strain by Clustal W method. The results showed that one sample was positive for p27 antigen by ELISA and IFA, which was named JS20 CR13. K subgroup primer PCR detected a product with a size of 560 bp;the total length of the provirus genome was 7 481 bp, in which the size of the env gene was 1 617 bp(gp85 gene being 1 008 bp, encoding 336 amino acids;gp37 gene being 609 bp, encoding 203 amino acids). Compared with the reference strains, the homology of the gp85 gene sequence of the JS20 CR13 isolate and the K subgroup reference strain was 94.0%-99.0%, which was significantly higher than that of the other subgroups;the amino acid sequence encoded by the gp85 gene belonged to the same evolutionary branch as the reference strain virus that had been identified as ALV-K, but had a long genetic evolutionary distance from other subgroups. The gp37 gene sequence of the JS20 CR13 isolate had the highest homology with the ALV-K reference strain JS14 CZ01, reaching 99.0%. The results showed that the isolated strain JS20 CR13 belonged to the same branch as the reference strain that had been previously identified as ALV-K, and the isolated strain was subgroup K ALV.

关 键 词：K亚群禽白血病病毒 崇仁麻鸡 遗传进化树 GP85基因 gp37基因 

分 类 号：S852.659.3[农业科学—基础兽医学]