Construction and identification of a recombinant adenovirus encoding fusion proteins of multi-stage antigens of Mycobacterium tuberculosis
WANG Li-mei1, JIANG Hong2, KANG Jian1, JIN Qian-qian1
1. Department of Microbiology and Pathogenic Biology, School of Basic Medicine, Air Force Medical University, Xi'an 710032, China; 2. The Center of Diagnosis and Treatment of Infectious Diseases, The Second Affiliated Hospital, Air Force Medical University, Xi'an 710038, China;
Abstract：This study aimed to construct a recombinant adenovirus encoding two fusion proteins, Ag85B-ESAT6(AE) and Rv2031c-Rv2626c(R2), from multi-stage antigens of Mycobacterium tuberculosis to facilitate research on a new vaccine against tuberculosis. The human Elongation Factor 1α (EF1α) promoter DNA sequence was synthesized in vitro and cloned into the adenovirus shuttle vector pAdTrack-CMV to construct an adenovirus shuttle vector containing CMV and EF1α double promoters. Previously constructed fusion protein genes of AE and R2 were amplified by PCR and cloned downstream of the CMV and EF1α promoters in the above adenovirus shuttle vector. The positive recombinant shuttle plasmid was named pAd-AE-R2. pAd-AE-R2 and the skeleton plasmid were co-transfected into HEK293 cells to obtain a recombinant adenovirus, denoted Ad-AE-R2. The gene and protein of AE and R2 expressed by Ad-AE-R2 were identified by RT-PCR and indirect immunofluorescence. IFA results showed that AE and R2 fusion protein genes were transcribed specifically in HEK293 cells, and specific green fluorescence was detected by monoclonal antibodies against Ag85B, ESAT-6, Rv2626c or Rv2031c in macrophages infected with Ad-AE-R2 by IFA, thus indicating that Ad-AE-R2 enabled correct expression of AE and R2 fusion proteins in host cells. In conclusion, the recombinant adenovirus encoding fusion proteins of AE and R2 from multi-stage antigens of Mycobacterium tuberculosis was successfully constructed, thus providing a research foundation for its use as a new TB vaccine.
王丽梅, 姜泓, 康健, 靳芊芊. 结核分枝杆菌抗原融合蛋白Ag85B-ESAT6和Rv2031c-Rv2626c重组腺病毒的构建及鉴定[J]. 中国人兽共患病学报, 2022, 38(3): 191-195.
WANG Li-mei, JIANG Hong, KANG Jian, JIN Qian-qian. Construction and identification of a recombinant adenovirus encoding fusion proteins of multi-stage antigens of Mycobacterium tuberculosis. Chinese Journal of Zoonoses, 2022, 38(3): 191-195.
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2022, Vol. 38 
