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表达狂犬病毒糖蛋白抗原的重组犬2型腺病毒的构建及鉴定
引用本文:赵忠鹏,夏咸柱,扈荣良,谢之景,闫芳.表达狂犬病毒糖蛋白抗原的重组犬2型腺病毒的构建及鉴定[J].微生物学报,2007,47(2):335-339.
作者姓名:赵忠鹏  夏咸柱  扈荣良  谢之景  闫芳
作者单位:军事医学科学院军事兽医研究所,长春,130062
基金项目:军队重点项目;国家科技攻关计划
摘    要:为研制一种预防犬科动物狂犬病的新型疫苗,将含有狂犬病毒ERA株糖蛋白基因(Rabies glycoprotein,Rgp)表达盒的穿梭质粒pVAXΔE3Rgp中的Rgp表达盒克隆入犬2型腺病毒(Canine adenovirus type2,CAV2)骨架质粒pPoly2-CAV2中,获得重组质粒pPoly2-CAV2-ΔE3-Rgp,释放其基因组,转染MDCK细胞系,获得E3缺失区(Deletion of early protein3,ΔE3)含有Rgp表达盒的重组病毒CAV2-ΔE3-Rgp。该重组病毒能在MDCK细胞上产生典型的腺病毒细胞病变。通过酶切、PCR、基因测序,表明该重组病毒含有完整的Rgp表达盒。通过RT-PCR、Western blot等检测,表明该重组病毒能够表达Rgp抗原。用该重组病毒免疫犬,3次免疫后,可以诱导犬产生特异的抗CAV2HI抗体,其效价超过1∶256和抗狂犬病病毒(Rabies virus,RV)中和抗体,其效价超过0.50IU/mL。试验结果表明,获得的重组病毒免疫犬后,能够产生抗狂犬病毒和腺病毒的高效价保护性抗体,是一种有潜力的犬科动物狂犬病毒腺病毒二联疫苗候选株。

关 键 词:狂犬病毒  犬2型腺病毒
文章编号:0001-6209(2007)02-0335-05
收稿时间:2006/7/10 0:00:00
修稿时间:2006-07-10

Construction and identification of recombinant canine adenovirus type 2 expressing exogenous rabies glycoprotein (Rgp)
ZHAO Zhong-peng,XIA Xian-zhu,HU Rong-liang,XIE Zhi-jing and YAN Fang.Construction and identification of recombinant canine adenovirus type 2 expressing exogenous rabies glycoprotein (Rgp)[J].Acta Microbiologica Sinica,2007,47(2):335-339.
Authors:ZHAO Zhong-peng  XIA Xian-zhu  HU Rong-liang  XIE Zhi-jing and YAN Fang
Institution:Institute of Military Veterinary; Academy of Military Medical Science; Changchun 130062; China;Institute of Military Veterinary; Academy of Military Medical Science; Changchun 130062; China;Institute of Military Veterinary; Academy of Military Medical Science; Changchun 130062; China;Institute of Military Veterinary; Academy of Military Medical Science; Changchun 130062; China;Institute of Military Veterinary; Academy of Military Medical Science; Changchun 130062; China
Abstract:Safe and effective vaccination is important for rabies prevention. Here, genetically engineered rabies vaccine CAV2-deltaE3-Rgp was developed and characterized. The recombinant genome pPoly2-CAV2-deltaE3-Rgp carrying the rabies glycoprotein (Rgp) cDNA was generated by a series of strictly gene cloning steps and infectious recombinant virus CAV2-deltaE3-Rgp was obtained by transfecting the recombinant genome into a canine kidney cell line, MDCK. To efficiently construct cloned recombinant canine adenovirus type 2 genome pPoly2-CAV2-deltaE3-Rgp bearing exogenous Rgp gene, The Rgp gene was first subcloned from the clone vector pMD18-T into the eukaryon expression vector pVAX1. The Rgp expression cassette was then subcloned into the shuttle vector pVAXdeltaE3 and subsequently into the canine adenovirus type 2 backbone vector pPoly2-CAV2. To indirectly confirm pPoly2-CAV2-deltaE3-Rgp, conventional restriction endonuclease digestion was performed. CAV2-deltaE3-Rgp can generate typical CPE of CAV-2. CAV2-deltaE3-Rgp was tested by restriction endonuclease digestion, PCR, DNA sequencing. As a result, The Rgp expression cassette was successfully integrated into the target region of the CAV2 genome. It is confirmed by RT-PCR, Western blot that CAV2-deltaE3-Rgp can express Rgp antigen in MDCK cell. This recombinant virus, CAV2-deltaE3-Rgp, was intramuscularly injected into dogs. All vaccinated dogs produced effective antibodies against CAV and RV after three inoculations. This recombinant virus would be prospective in immunizing dogs against CAV and RV.
Keywords:rabies  Canine adenovirus type 2(CAV-2)
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