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Construction and Immunological Properties of DNA Vaccine of Mycobacterium tuberculosis

Author: FanXiongLin
Tutor: XuZhiKai
School: Fourth Military Medical University
Course: Pathogen Biology ( Molecular Microbiology )
Keywords: Mycobacterium tuberculosis Ag85B DNA vaccine DNA immunization gene transfection RT-PCR PCR adoptive immunity
CLC: R392
Type: PhD thesis
Year: 2001
Downloads: 217
Quote: 1
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Abstract


Tuberculosis(TB) is a chronic respiratory infectious disease caused by the pathogen Mycobacterium fuberculosis. TB remains an important global public health problem, because about one-third of the world’s populations are infected with the M. tuberculosis and there are 10 million new cases and 3 million deaths annually, in recent years, such factors as increasing frequency of drug-resistance M tuberculosis isolates and incidence of HI V-associated tuberculosis and increasement of population movement, have aggravated further the opportunity of people worldwide face to the threat of TB. Mycobacterium bovis BCG is the only tuberculosis vaccine available for human use in many developing countries, yet its protection efficacy varies greatly. Another limitation is that it interrupts the diagnosis of TB by using purified protein derivative (PPD). Furthermore, the live BCG vaccine represents a potential health risk to immunocompromized individuals. It should be very important to develop a more effective vaccine of TB. It has been proved that the administration of DNA vaccines could generate both hwnoral and cell-mediated immune responses in many animal models of infectious diseases including TB. 24 mycobacterial antigens 6 mostly from cultural filtration proteins(CFP) of Mtuberculosis have been expressed using plasmid DNA eukaryotic vector. Among these, several antigens have showed higher protection efficacy in animal models such as Ag85 complex, ESAT6, MPT64, Hsp65 and PstS3, etc. Thus, DNA vaccination could be a promising effective solution to protect against TB. It has been showed that over 800 proteins result in the CFP of Mtuberculosis. Important methods used to identify protective antigens from CFP include, that proteins separated by biochemistry methods could be used to stimulate lymphocytes and detect kinds and quantity of cytokines secreted by lymphocytes; and that sera of patients with TB or mice infected with Mtuberculosis could be used to screen expression library of Mtuberculosis. The development of DNA vaccine of Mtuberculosis could be a potential way to identify protective antigens in vivo. In this paper, we want to develop new vaccine against TB and search for a rapid, effective and inexpensive model of DNA vaccination to screen protective antigens. The gene encoding the Ag85B mature form with two amino acids of signal sequence from genome of M tuberculosis H37Ra strain was amplified by PCR., and inserted it into cloning vector pUC 19 after restriction endonuclease digestion. Gene fragment encoding Ag85B mature protein was correctly inserted into the vector as confirmed by partial nucleotide sequencing and restriction endonuclease digestion, and then was subcloned to sites cut with HindIIl plus EcoR I of eukaryotic expression vector pcDNA3(Invitrogen Corp.) under the control of the CMV promoter. The cloning gene was correctly inserted into the vector pcDNA3 as also confirmed by restriction endonuclease digestion. This construction was called pTB3Om. Sequence of Ag85B mature protein was compared by Blast homological search via GenBank. It shows that nucleotide sequence of Ag85B mature protein of Mtuberculosis I-137Ra strain has the highest degree of homology with that of corresponds of M tuberculosis complexes and 7 higher degree of similarity with that of corresponding proteins of non-M tuberculosis. Nucleotide sequence

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