山羊地方性鼻内肿瘤病毒全基因组序列分析及其gag基因促肿瘤发生的机制研究

doi:10.11843/j.issn.0366-6964.2021.011.018

摘要/Abstract

摘要： 本研究旨在获得山羊地方性鼻内肿瘤病毒（enzootic nasal tumor virus 2，ENTV-2）全基因组序列并进行分析。从福建某羊场的山羊鼻内肿瘤组织取样，针对ENTV-2设计引物，通过RT-PCR方法从肿瘤组织中分段扩增出6个特异性产物，测序后利用生物学软件DNAStar进行拼接，获得长度为7 443 bp全基因组序列，将其命名为ENTV-2-FJ。ENTV-2-FJ基因组结构与其他逆转录病毒类似，具有5'-U5-gag-pro-pol-env-U3-3'典型结构，包含4个开放阅读框和侧翼非编码区以及末端重复序列。为制备兔抗ENTV-2 gag蛋白多克隆抗体，将特异性扩增的gag基因克隆至pET-21a （+）载体，构建重组质粒pET-21a-gag，鉴定后转化至BL21（DE3）细胞并经IPTG诱导成功表达分子量约为70 ku的融合蛋白。Western blot分析表明，制备的兔抗gag蛋白抗体能在患病山羊肿瘤组织和表达gag基因的细胞中特异性地检测出gag蛋白。为了深入研究ENTV-2-FJ致瘤机制，作者构建了过表达gag基因的K562细胞系，并进行裸鼠致瘤试验。结果显示， gag蛋白通过激活JAK2-STAT5信号通路促进肿瘤的生长。综上，本研究获得了ENTV-2-FJ全基因组序列并对其进行分析，制备并验证了gag蛋白的多克隆抗体，揭示了gag蛋白的作用机制，这些结果为阐明ENTV-2的致病机制提供了科学依据。

关键词: 山羊地方性鼻内肿瘤病毒, 全基因组序列, gag, 多克隆抗体制备, 致病性研究

Abstract: In order to obtain and analyze the complete genome sequences of enzootic nasal tumor virus 2 (ENTV-2) in goat intranasal tumor tissues obtained from a Fujian farm, six specific fragments of the ENTV-2 were amplified from these tumor tissues by RT-PCR. The products were then sequenced and assembled using biological software DNAStar to obtain the whole genome sequences. We found that whole length of the viral genome was 7 443 bp, and the virus was named as ENTV-2-FJ. The structure of ENTV-2-FJ genome is similar to other retroviruses, which has a 5'-U5-gag-pro-pol-env-U3-3' canonical structure with four open reading frames, flanked by non-coding regions and terminal repeats sequence. In order to prepare rabbit anti-ENTV-2 gag protein polyclonal antibody, a pair of specific primers was designed and used to clone the gag gene that was then ligated to pET-21a (+) vector. The recombinant plasmid pET-21a-gag was constructed, transferred into BL21 (DE3) competent cells, and the recombinant fusion protein with a molecular weight of about 70 ku was successfully induced by IPTG. Western blot analysis showed that the prepared rabbit anti-gag protein antibody specifically detected the gag protein in the tumor tissues in diseased goats and cells expressing gag gene. Furthermore, we generated K562 cell lines overexpressing the gag gene and conducted tumorigenic experiments on nude mice. The results showed that overexpression of gag gene promoted tumor growth through activation of JAK2-STAT5 signaling pathway. Taken together, this study provides the complete genomic sequences of ENTV-2-FJ and rabbit polyclonal antibodies against viral gag protein, and reveals an important role of the gag protein in tumorigenesis mediated by ENTV-2-FJ.

Key words: ENTV-2, whole gene sequence, polyclonal antibody preparation, gag, pathogenicity research

中图分类号:

S857.4

潘启东, 曾显成, 杨彬偲, 刘庆华, 徐泉明, 陈吉龙. 山羊地方性鼻内肿瘤病毒全基因组序列分析及其gag基因促肿瘤发生的机制研究[J]. 畜牧兽医学报, 2021, 52(11): 3165-3174.

PAN Qidong, ZENG Xiancheng, YANG Bincai, LIU Qinghua, XU Quanming, CHEN Jilong. Analysis of Complete Genomic Sequences of Enzootic Nasal Tumor Virus 2 and Role of Its gag Gene in Tumorigenesis[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(11): 3165-3174.

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