猪附红细胞体ENO基因重组腺病毒穿梭质粒的构建及真核表达

doi:10.16431/j.cnki.1671-7236.2018.08.009

摘要/Abstract

摘要：

为构建猪附红细胞体ENO基因重组腺病毒穿梭质粒，试验根据GenBank中登录的猪附红细胞体ENO基因序列（登录号：CP002525.1）设计特异性引物，对ENO基因进行PCR扩增，并将纯化后的PCR产物克隆到pMD19-T载体中。用Kpn Ⅰ和Xho Ⅰ对pMD19T-ENO进行双酶切后，将其亚克隆至腺病毒穿梭载体PCR259中，构建PCR259-ENO重组质粒，提取重组质粒进行鉴定。应用脂质体介导转染法将鉴定正确的PCR259-ENO重组穿梭质粒转染293细胞，应用间接免疫荧光法（IFTA）检测ENO基因在293细胞中的表达。结果显示，试验克隆的ENO基因长为1 182 bp，编码393个氨基酸，与GenBank中ENO基因序列（登录号：CP002525.1）同源性为99%。构建的重组腺病毒穿梭质粒PCR259-ENO经PCR和酶切鉴定正确，并且能在293细胞中表达，表明ENO基因成功插入腺病毒穿梭质粒PCR259中，重组腺病毒穿梭质粒PCR259-ENO构建成功。

关键词: 猪附红细胞体; ENO基因; 重组腺病毒穿梭质粒; 表达

Abstract:

To construct recombinant adenovirus shuttle plasmid of ENO gene of Mycoplasma suis and express it in eukaryotic cells,a pair of primers was designed and synthesized according to the ENO gene sequence of Mycoplasma suis in GenBank (accession No.:CP002525.1) in this study.ENO gene was amplified by PCR method,and the products were cloned into the pMD19-T vector.The pMD19T-ENO plasmid was digested with Kpn Ⅰ and Xho Ⅰ, and then subcloned into the adenovirus shuttle vector PCR259.The PCR259-ENO plasmid was constructed and then identified by PCR and enzyme digestion.The PCR259-ENO recombinant shuttle plasmid was transfected into 293 cells by liposome-mediated transfection,and the expression of ENO gene in 293 cells was detected by IFTA technology.The results showed that the cloned ENO gene was 1 182 bp in length and encoded 393 amino acids with a homology of 99% with the ENO gene sequence in GenBank (accession No.:CP002525.1).The constructed recombinant adenovirus shuttle plasmid PCR259-ENO was identified by PCR and restriction enzyme digestion correctly, and was able to be expressed in 293 cells,indicating that ENO gene was successfully inserted into adenovirus shuttle plasmid PCR259 and the recombinant shuttle plasmid was successfully constructed.

Key words: Mycoplasma suis; ENO gene; recombinant adenovirus shuttle plasmid; expression

中图分类号:

Q784

王淼, 倪婷婷, 伍生军, 赵云, 宋建臣, 董亮, 薛书江. 猪附红细胞体ENO基因重组腺病毒穿梭质粒的构建及真核表达[J]. 《中国畜牧兽医》, 2018, 45(8): 2113-2118.

WANG Miao, NI Tingting, WU Shengjun, ZHAO Yun, SONG Jianchen, DONG Liang, XUE Shujiang. Construction and Eukaryotic Expression of Recombinant Adenovirus Shuttle Plasmid of Mycoplasma suis ENO Gene[J]. , 2018, 45(8): 2113-2118.

参考文献

[1] 杨卉新,单思,巴彩凤.猪附红细胞体病研究进展[J].现代畜牧兽医,2013,5:48-52.YANG H X,SHAN S,BA C F.Research progress of Mycoplasma suis[J].Modern Journal of Animal Husbandry and Veterinary Medicine,2013,5:48-52.(in Chinese)
[2] 王海军,王兵,任士飞,等.犬附红细胞体病研究进展[J].今日畜牧兽医,2017,10:36.WANG H J,WANG B,REN S F,et al.Research progress of dog Eperythrozoon[J].Today Animal Husbandry and Veterinary Medicine,2017,10:36.(in Chinese)
[3] 詹海杰,侯志宏,王国艳,等.猪附红细胞体病鉴别诊断研究进展[J].山西农业科学,2014,42(1):97-99.ZHAN H J,HOU Z H,WANG G Y,et al.Progress in differential diagnosis of Mycoplasma suis[J].Shanxi Agricultural Science,2014,42(1):97-99.(in Chinese)
[4] 杨孟鸿,游佳斌.猪附红细胞体病诊治进展[J].中兽医学杂志,2017,1:98-99.YANG M H,YOU J B.Progress in diagnosis and treatment of Mycoplasma suis[J].Chinese Journal of Traditional Veterinary Science,2017,1:98-99.(in Chinese)
[5] KANEGAE Y,LEE G,SATO Y,et al.Efficient gene activation in mammalian cells by using recombinant adenovirus expressing site-specific Cre recombinase[J].Nucleic Acids Research,1995,23(19):3816-3821.
[6] 王晓蕾,胡小然.猪附红细胞体病的研究进展[J].中国畜禽种业,2014,10(8):117-118.WANG X L,HU X R.Progress of research on Mycoplasma suis[J].The Chinese Livestock and Poultry Breeding,2014,10(8):117-118.(in Chinese)
[7] 韩雨,云升.附红细胞体感染对机体免疫功能影响的研究进展[J].医学动物防制,2016,32(1):57-60.HAN Y,YUN S.Research progress on the influence of Eperythrozoon infection on the immune function of the body[J].Journal of Medical Pest Control,2016,32(1):57-60.(in Chinese)
[8] 唐丽,李晓云,贾杏林.猪附红细胞体分子生物学研究进展[J].中国畜牧兽医,2013,40(7):81-83.TANG L,LI X Y,JIA X L.Advances in molecular biology of Eperythrozoon suis[J].China Animal Husbandry & Veterinary Medicine,2013,40(7):81-83.(in Chinese)
[9] 郭焕平,贾立军,于龙政,等.犬新孢子虫NcAMA1基因重组腺病毒穿梭质粒的构建及其真核表达[J].中国兽医科学,2014,44(3):282-286.GUO H P,JIA L J,YU L Z,et al.Construction of recombinant adenovirus vector of NcAMA1 gene of canine conidia gene and its eukaryotic expression[J].Chinese Veterinary Science,2014,44(3):282-286.(in Chinese)
[10] 汪东篱,姚炜,郑庆鸣,等.一起腺病毒引起的急性呼吸道感染暴发疫情的调查研究[J].病毒学报,2017,6:920-925.WANG D L,YAO W,ZHENG Q M,et al.An outbreak of acute respiratory infection caused by adenovirus[J].Chinese Journal of Virology,2017,6:920-925.(in Chinese)
[11] BRUNA-ROMERO O,ROCHA C D,TSUJI M,et al.Enhanced protective immunity against malaria by vaccination with a recombinant adenovirus encoding the circumsporozoite protein of Plasmodium lacking the GPI-anchoring motif[J].Vaccine,2004,22:3575-3584.
[12] JIANG J,ZHANG Y,PENG K,et al.Combined delivery of a TGF-β inhibitor and an adenoviral vector expressing interleukin-12 potentiates cancer immunotherapy[J].Acta Biomaterialia,2017,61:114-123.
[13] 张影.猪附红细胞体α-烯醇化酶基因的表达及间接ELISA检测方法的建立[D].延吉:延边大学,2013.ZHANG Y.Expression of α-enolase gene in Eperythrozoon suis and establishment of indirect ELISA detection method[D].Yanji:Yanbian University,2013.(in Chinese)
[14] HOELZLE L E.Significance of haemotrophic Mycoplasmas in veterinary medicine with particular regard to the Mycoplasma suis infection in swine[J].Berliner und Munchener Tierarztliche Wochenschrift, 2007,120(1-2):34-41.
[15] GROEBEL K,HOELZLE K,WITTENBRINK M M,et al.Mycoplasma suis invades porcine erythrocytes[J].Infection and Immunity,2009,77(2):576-584.
[16] 李书光,唐延平.猪附红细胞体病研究进展[J].中国畜牧兽医,2008,35(10):108-110.LI S G,TANG Y P.Progress in the study of Mycoplasma suis[J].China Animal Husbandry & Veterinary Medicine,2008,35(10):108-110.(in Chinese)
[17] 张影,钱年超,贾立军,等.猪附红细胞体DnaJ基因的克隆及真核表达[J].中国兽医科学,2013,1:65-69.ZHANG Y,QIAN N C,JIA L J,et al.Cloning and eukaryotic expression of DnaJ gene from Eperythrozoon suis[J].Chinese Veterinary Science,2013,1:65-69.(in Chinese)
[18] SCHREINER S A,SOKOLI A,FELDER K M,et al.The surface-localised α-enolase of Mycoplasma suis is an adhesion protein[J] Veterinary Microbiology,2012,156(1-2):88-95.
[19] 何婉婉,周立,刘涛,等.溶瘤腺病毒作为癌症基因治疗载体的研究进展及安全性评价[J].中国细胞生物学学报,2013,35(9):1386-1391.HE W W,ZHOU L,LIU T,et al.Research progress and safety evaluation of adenovirus as a vector for gene therapy of cancer[J].Chinese Journal of Cell Biology,2013,35(9):1386-1391.(in Chinese)
[20] 李秀博,崔尚金,王春仁,等.犬腺病毒作为载体在疾病治疗和预防中的作用研究进展[J].现代畜牧兽医,2017,9:54-58.LI X B,CUI S J,WANG C R,et al.Progress on the role of canine adenovirus as vector in the treatment and prevention of disease[J].Modern Journal of Animal Husbandry and Veterinary Medicine,2017,9:54-58.(in Chinese)
[21] 杨勇,周东明.腺病毒载体疫苗的临床研究进展[J].生命的化学,2014,34(1):46-51.YANG Y,ZHOU D M.Progress in clinical research of adenoviral vector vaccine[J].Chemistry of Life, 2014,34(1):46-51.(in Chinese)
[22] FOUGEROUX C,HOLST P J.Future prospects for the development of cost-effective adenovirus vaccines[J].International Journal of Molecular Sciences,2017,18(3):686.
[23] 王勇,杨侃侃,王元红,等.表达羊口疮病毒B2L基因重组腺病毒的构建及鉴定[J].扬州大学学报(农业与生命科学版),2017,38(3):6-10.WANG Y,YANG K K,WANG Y H,et al.Construction and identification of recombinant adenovirus expressing B2L gene of sheep mouth sore virus[J].Journal of Yangzhou University (Agriculture and Life Sciences Edition),2017,38(3):6-10.(in Chinese)