转乳铁蛋白肽和α干扰素基因的牛胎儿成纤维细胞的制备

畜牧兽医学报

转乳铁蛋白肽和α干扰素基因的牛胎儿成纤维细胞的制备

余大为¹，张守峰²，朱化彬¹，张锦霞²，范宗兴¹，郝海生¹，刘岩¹，赵学明¹，秦彤¹，杜卫华^1*

(1.中国农业科学院北京畜牧兽医研究所，北京 100193； 2.中国军事医学科学院军事兽医研究所，长春 130062)

出版日期:2012-10-25 发布日期:2012-10-25
通讯作者: 杜卫华，E-mail: dwh@iascaas.net.cn
作者简介:余大为（1986-），男，重庆人，硕士，主要从事动物胚胎工程技术研究，Tel：010-62815691, E-mail：ydw023@163.com
基金资助:
转基因重大专项（2009ZX08007-007B）

Construction of Lactoferricin and Interferon-α Genes Transgenic Bovine Fetal Fibroblasts

YU Da-wei¹，ZHANG Shou-feng²，ZHU Hua-bin¹，ZHANG Jin-xia²，FAN Zong-xing¹，HAO Hai-sheng¹，LIU Yan¹，ZHAO Xue-ming¹，QIN Tong¹，DU Wei-hua^1*

(1. Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193，China;
2. Institute of Military Veterinary，Academy of Military Medical Science，Changchun 130062，China）

Online:2012-10-25 Published:2012-10-25

摘要/Abstract

摘要： 旨在构建含有乳铁蛋白肽基因和α干扰素基因的载体，并制备转基因牛胎儿成纤维细胞，为研制抗乳房炎和口蹄疫转基因克隆牛提供供体细胞。本研究构建了由山羊β-酪蛋白启动子启动的牛乳铁蛋白肽基因和由CMV启动子调控的人α干扰素基因的载体，以EGFP基因作为报告基因， neo基因作为筛选基因；分别采用脂质体和电击法转染牛胎儿成纤维细胞， G418筛选得到转基因阳性细胞，并用PCR方法检测转基因细胞中目的基因的整合情况，用Western blotting检测α干扰素蛋白的表达。结果，得到了同时含有牛乳铁蛋白肽基因和人α干扰素基因的转基因牛胎儿成纤维细胞，并且α干扰素蛋白在转基因细胞中能有效表达。

Abstract: The aim of this study was to construct expression vector containing Lactoferricin gene and interferon-α gene which was transfected into the bovine fetal fibroblasts. The transgenic cells got would be used as the donor cells for SCNT to produce the transgenic bovine that would resist mastitis disease and FMD. The vector with a bovine Lactoferrincin B cassette containing a goat β-casein regulatory sequence, a human interferon-α cassette containing the immediate early promoter of CMV, the EGFP gene as reporter and the neo gene as positive selection marker, was constructed. FuGENE Transfection Reagent and Amaxa Nucleofector were used in transfection. After 48 hours，the cells were selected with G418 and detected by PCR. The expression of interferon-α protein was detected by Western blotting. The transgenic bovine fetal fibroblast cell lines were constructed in which Lactoferricin and interferon-α genes were integrated and the expression of interferon-α protein was detected.

中图分类号:

S823
S814.8

余大为，张守峰，朱化彬，等. 转乳铁蛋白肽和α干扰素基因的牛胎儿成纤维细胞的制备[J]. 畜牧兽医学报, doi: .

YU Da-wei，ZHANG Shou-feng，ZHU Hua-bin，et al. Construction of Lactoferricin and Interferon-α Genes Transgenic Bovine Fetal Fibroblasts[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, doi: .

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参考文献

［1］王晓铄, 俞英.表观遗传对炎症的调控机制及其在奶牛乳房炎抗病育种中的应用前景［J］.遗传,2010,32(7): 663-669.

［2］DONOVAN D M, KERR D E, WALL R J.Engineering disease resistant cattle［J］.Transgenic Res,2005,14(5): 563-567.

［3］常智双, 窦同喜, 腾井华.牛口蹄疫的诊断与防控［J］.畜牧与饲料科学,2010,31(3): 174-175.

［4］余大为, 杜卫华, 朱化彬.家畜转基因育种研究进展［J］.遗传,2011,33(5): 459-468.

［5］STAEHELI P.Intracellular immunization: a new strategy for producing disease-resistant transgenic livestock? ［J］.Trends Biotechnol,1991,9(3): 71-72.

［6］COOPER C A, BRUNDIGE D R, REH W A, et al.Lysozyme transgenic goats' milk positively impacts intestinal cytokine expression and morphology［J］.Transgenic Res,2011,20(6): 1235-1243.

［7］MAGA E A, CULLOR J S, SMITH W, et al.Human lysozyme expressed in the mammary gland of transgenic dairy goats can inhibit the growth of bacteria that cause mastitis and the cold-spoilage of milk［J］.Foodborne Pathog Dis,2006,3(4): 384-392.

［8］LIU Y F, HAN F F, XIE Y G, et al.Comparative antimicrobial activity and mechanism of action of bovine lactoferricin-derived synthetic peptides［J］.Biometals,2011,24(6): 1069-1078.

［9］AHMED C M, WILLS K N, SUGARMAN B J, et al.Selective expression of nonsecreted interferon by an adenoviral vector confers antiproliferative and antiviral properties and causes reduction of tumor growth in nude mice［J］.J Interferon Cytokine Res,2001,21 (6):399-408.

［10］RUTHERFORD M N, KUMAR A, COULOMBE B, et al.Expression of intracellular interferon constitutively activates ISGF3 and confers resistance to EMC viral infection［J］. J Interferon Cytokine Res,1996,16(7): 507-510.

［11］张锦霞, 张守峰, 郭学军, 等.抗菌肽重组逆转录病毒载体的构建、重组病毒的包装及其在牛奶中的稳定表达［J］.中国兽医学报,2008,28(12): 1433-1437.

［12］HARLE P, CULL V, GUO L, et al.Transient transfection of mouse fibroblasts with type I interferon transgenes provides various degrees of protection against herpes simplex virus infection［J］.Antiviral Res,2002,56(1): 39-49.

［13］YEOW W S, LAWSON C M, BEILHARZ M W.Antiviral activities of individual murine IFN-alpha subtypes in vivo: intramuscular injection of IFN expression constructs reduces cytomegalovirus replication［J］.J Immunol,1998,160(6): 2932-2939.

［14］MEAGER A.Biological assays for interferons［J］.J Immunol Methods,2002,261(1-2): 21-36.

［15］WILL A, HEMMANN U, HORN F, et al.Intracellular murine IFN-gamma mediates virus resistance, expression of oligoadenylate synthetase, and activation of STAT transcription factors［J］.J Immunol,1996,157(10): 4576-4583.

［16］TAKAHASHI M, TAKAHASHI H, HAMANO S, et al.Possible role of interferon-tau on in vitro development of bovine embryos［J］.J Reprod Dev,2003,49(4): 297-305.

［17］刘中华, 王华岩, 乔宪凤, 等.转基因技术在动物遗传改良上的应用进展［J］.湖北农业科学,2009,48(2): 481-486.

［18］芦小燕, 解慧琪, 李莉,等.人成纤维细胞稳定转染的方法比较［J］.四川大学学报,2008,39(4): 654-657.

［19］崔奎青, 刘庆友, 汤刚彬, 等.影响水牛胎儿成纤维细胞转染效率的因素［J］.中国畜牧杂志,2008,44(19): 16-19.

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