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| Construction and Selection of the Recombinant Fowlpox Expressing HIV-1 gag |
| ZHU Yi-long1, LI Chang2, GUO Yan1, LIU Cun-xia2, DU Shou-wen2, WANG Mao-peng2, JIN Ning-yi2 |
1. Changchun University of Traditional Chinese Medicine, Changchun 130117, China;
2. Key Laboratory of Jilin Province for Zoonosis Prevention and Control, Genetic Engineering Laboratory of PLA, Academy of Military Medical Sciences of PLA, Changchun 130122, China |
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Abstract To construct and select expressing HIV-1 gag protein in recombinant fowlpox virus. Primers were designed and synthesized. HIV-1 gag was amplified by PCR and ligated with pMD 18-T Sample Vector. The results sequence of HIV gag was correct, and then it was inserted into pTKETwhich was made in our laboratory to construct the recombination shuttle plasmid pTKET-HIV gag. The plasmid pTKET-HIV gag and 282E4 strain fowlpox virus were co-transfected 80% confluent Chicken Embryo Fibroblast (CEF) cells to select the recombinat fowlpox virus with EGFP as the reporter gene. The recombination fowlpox virus was verified and analyzed by PCR, RT-PCR and Western blot.The gene has been integrated into recombinant fowlpox virus genome by PCR after 10 times plaques screening, and the results of RT-PCR and Western blot showed that HIV-1 gag was successfully expressed in infected cells. The recombinant fowlpox virus which was continuously passaged 20 times also showed exogenous genes integration, transcription and expression by PCR, RT-PCR and Western blot. FPV-TK was not amplified by PCR and showed that the HIV-1 gag gene has good genetic stability in recombinant fowlpox virus which has been purified.The recombinant fowlpox virus expressing HIV gag protein was successfully generated, which provide a good foundation for future immunity experiment.
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Received: 04 November 2013
Published: 25 January 2014
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