Construction Of Recombinant Bait Plasmids For Studing CMV 2b Protein Inteaction With Musa

Cucumber mosiac virus(CMV) is a kind of wide-spreading and prevalent plant RNA virus, which is competent to infect more than 100 families,1200 species of both monocotyledon and dicotyledon plants. It belongs to Bromoviridate, Cucumoviru. The genome of CMV was maked by +ssRNA, which contains RNA1, RNA2, RNA3 and the subgenome(RNA4), some of the samples also carry satellite RNAs. In a word, the genome structure of CMV is uncomplicated, thus is a typical model to research of the pathogenic mechanism of the RNA viruses. In recent years, many functions of 2b gene has been found, which maybe a key factor for CMV interaction with the host. The expression product of 2b gene, the 2b protein, is a suppressor of post-transcript gene silencing(PTGS). It may be the main reason of CMV infecting many hosts. In this paper,2b gene has been studied in following aspects.1 Constructing of BacterioMatch II Two-Hybrid System bait plasmid pBT-CMV 2b, testing of its expression and self-activation.BacterioMatch II Two-Hybrid System bait plasmid could be used to screen the proteins which could interact with 2b protein from BacterioMatch II Two-Hybrid System cDNA library, which maybe explain the functions of 2b protein.2b gene was obtained by PCR amplification, then was inserted into the pBT vector, which has been fused the full-length bacteriophageλcⅠrepressor protein, aquiring the pBT-CMV 2b recombinant plasmid. DNA sequencing results showed that the nucleotide sequences and reading frames of the CMV 2b coding region were correct, which means that the pBT-CMV 2b recombinant plasmid was constructed successfully. The bait plasmid, pBT-CMV 2b, was then transformed into the E.coli XLl-Blue MRF'report competent cell and cultured at 30℃and induced with IPTG The whole cell extracts were detected by SDS-PAGE and results showed that the CMV 2b fusion protein couldn't be directly detected by SDS-PAGE. However,λcⅠ-2b fusion protein was detected by Western bot. This result means thatλcⅠ-2b fusion protein was expressed in host cells. Recombinant bait plasmid pBT-CMV 2b and plasmid pTRG cotransformed into E.coli XLl-Blue MRF report strains competent cell, culturing on no 3-AT plate and 3-AT plate at 37℃respectively to observe the growth of colonies, and compared with the negative control. The results showed that the recombinant plasmid pBT-CMV 2b could not be self-activated and could be used for the library screening experiments.2 The preparation ofλcⅠpolyclonal antibody.λcⅠpolyclonal antibody was necessary for Western blot to validate BacterioMatch II Two-Hybrid System bait plasmid expression.λcⅠgene was obtained by PCR amplification, and then was cloned to the expression vector pET-32b, constructing the pET-32b-λcⅠrecombinant expression plasmid. DNA sequencing results showed that the nucleotide sequences and open reading frame were correct, which means that the pET-32b-λcⅠrecombinant plasmid was constructed successfully. Then the recombinant plasmid was transformed into the E.coli DE3 competent cells, induced by IPTG at 20℃for expressing soluble protein. The protein was purified by the Ni2+-NTA columns. The purified protein was mixed with the Freund's adjuvant, then injected into rabbits to preparation antiserum. The titre of the antiserum was proved to be 1:25000 by ID-ELISA and it was qualified to BacterioMatch II Two-Hybrid System to validate the expression of recombinant bait plasmid by Western blot.3 Yeast two-hybrid system recombinant bait plasmid construction and self-activation testing.Because of the different characteristics of Yeast two-hybrid system and BacterioMatch II Two-Hybrid System, Yeast Two-Hybrid System bait plasmid could be used to find different proteins which can interact with 2b protein from Yeast two-hybrid system cDNA library and the functions of 2b protein could be further explained by this way. CMV 2b gene was cloned to pDESTTM 32 by GatewayTM technology through the entry cloning vector which takes the adavantage of site-specific recombination properties of bacteriophage. Firstly, CMV 2b gene was cloning to entry cloning vector pDONRTM207 by BP recombination reactions, DNA sequencing results showed that recombinant plasimid pDONRTM207-CMV 2b was constructed successfully. pDONRTM207 and pDESTTM32 appeared same resistance in E.coli cells, so the recombinant plasmid pDESTTM32-CMV 2b could not be screened by conventional means. For a large number of pDONRTM207-CMV 2b recombination strains could grow on the resistent plate either.Fortunately, two specificly digestion site EcoRⅤand BglⅡwhich enable to inactivate the resistance site but not influenced LR recmbination reaction by its digestion were found by analyzed the pDONRTM207-CMV 2b sequences enzyme digest site. The recombinant pDESTTM32-CMV 2b bait plasmid obtained by LR recombination reactions with pDONRTM207-CMV 2b which digested by EcoRⅤand BglⅡ. DNA sequencing results showed that recombinant bait plasimid pDESTTM32-CMV 2b was constructed successfully.Recombinant bait plasmid pDESTTM32-CMV 2b and plasmid pDESTTM22 cotransformed into MaV203 competent cells, culturing on different concentration 3-AT plates at 37℃and observe the growth of colonies, comparing with the controls. Results showed that the recombinant bait plasmid pDESTTM32-CMV 2b had weak self-activation which could be restrained by 50-75mM 3-AT, So it could be used to the further library screening experiments under this conditions.