1株髓细胞瘤型J亚群禽白血病病毒感染性克隆的构建与病毒拯救

doi:10.11843/j.issn.0366-6964.2013.05.012

畜牧兽医学报 ›› 2013, Vol. 44 ›› Issue (5): 754-759.doi: 10.11843/j.issn.0366-6964.2013.05.012

1株髓细胞瘤型J亚群禽白血病病毒感染性克隆的构建与病毒拯救

林艳¹，夏静¹，邹年莉¹，郭明萍¹，王富妍¹，赵扬¹，文心田^1,2，曹三杰^1,2，黄勇^{1, 2*}

（1. 四川农业大学动物医学院，雅安 625014；2. 四川农业大学动物疫病与人类健康四川省重点实验室，雅安 625014）

收稿日期:2012-11-21 出版日期:2013-05-23 发布日期:2013-05-23
通讯作者: 黄勇，E-mail: hyong601@163.com，Tel: 0835-2886117
作者简介:林艳（1986-），女，羌族，四川绵阳人，硕士研究生，主要从事家禽传染病研究，E-mail：linyan102@126.com
基金资助:
教育部《长江学者和创新团队发展计划》创新团队项目（IRTO848）

Construction and Virus Rescue of Infectious Molecular Clones of Subgroup J Avian Myeloid Leukosis Virus

LIN Yan¹, XIA Jing¹, ZOU Nian-li¹, GUO Ming-ping¹, WANG Fu-yan¹, ZHAO Yang¹, WEN Xin-tian^{1, 2}, CAO San-jie^{1, 2}, HUANG Yong^{1, 2*}

(1. College of Veterinary Medicine, Sichuan Agricultural University, Ya’an 625014, China;2. Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Ya’an 625014, China)

Received:2012-11-21 Online:2013-05-23 Published:2013-05-23

摘要/Abstract

摘要：

为构建髓细胞瘤型J亚群禽白血病病毒（avian leukosis virus，ALV-J）SCGS-1株前病毒cDNA分子标记感染性克隆，根据SCGS-1全基因测序结果，分3段进行全序列PCR扩增，顺次连接至pUC19，构建SCGS-1株前病毒cDNA感染性克隆pUC-SCGS；通过重叠PCR方法对SCGS-1基因组进行沉默突变，在4 684位点引入SalⅠ位点，构建SCGS-1株分子标记感染性克隆pUC-△SCGS；以pUC-SCGS和pUC-△SCGS重组质粒转染CEF进行病毒拯救，并通过PCR、间接免疫荧光与双抗体夹心ELISA进行拯救病毒检测。结果显示，成功构建pUC-SCGS与pUC-△SCGS重组质粒，转染后盲传第3代、第4代细胞与上清中均检测到拯救病毒；间接免疫荧光与抗原ELISA方法分别在CEF细胞和上清中检测到ALV-J抗原。成功拯救获得分子标记ALV-J。

关键词: 髓细胞瘤型, J亚群禽白血病病毒, 感染性克隆, 分子标记, 病毒拯救

Abstract:

The objective of this study was to construct the infectious molecular clone with molecular marker of subgroup J avian myeloid leukosis virus (ALV-J) strain SCGS-1. A full-length infectious clone of ALV-J (pUC-SCGS) was constructed by cloning and combining of three fragments using PCR method from SCGS-1. SalⅠ site was introduced on 4684nt of SCGS-1 by overlapping PCR to form another infectious clone and named pUC-△SCGS. The two plasmids, pUC-SCGS and pUC-△SCGS, were transfected into CEF, and the rescued viruses were detected by PCR, avian leukosis virus antigen test kit and indirect immunofluorescence assay (IFA). Digestion and sequence analysis revealed that the infectious clone pUC-SCGS and pUC-△SCGS were constructed correctly. PCR, ELISA test and IFA results showed that the 3rd and 4th generation of rescued virus were positive, while the controlled CEF were negative. Rescued virus and the virus with molecular marker of subgroup J avian myeloid leukosis virus were successfully constructed, named rSCGS-1 and r△SCGS-1.

Key words: myeloid leukosis, subgroup J avian leukosis virus, infectious molecular clone, molecular marker, virus rescue

中图分类号:

S852.659.3

林艳，夏静，邹年莉，郭明萍，王富妍，赵扬，文心田，曹三杰，黄勇. 1株髓细胞瘤型J亚群禽白血病病毒感染性克隆的构建与病毒拯救[J]. 畜牧兽医学报, 2013, 44(5): 754-759.

LIN Yan, XIA Jing, ZOU Nian-li, GUO Ming-ping, WANG Fu-yan, ZHAO Yang, WEN Xin-tian, CAO San-jie, HUANG Yong. Construction and Virus Rescue of Infectious Molecular Clones of Subgroup J Avian Myeloid Leukosis Virus[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2013, 44(5): 754-759.

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1株髓细胞瘤型J亚群禽白血病病毒感染性克隆的构建与病毒拯救

Construction and Virus Rescue of Infectious Molecular Clones of Subgroup J Avian Myeloid Leukosis Virus

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