The Preliminary Research On The Receptor Of IBDV Gt VP2 In CEF By The Yeast Two-Hybrid System

Infectious bursal disease virus(IBDV),the causative agent of infectious bursal disease(IBD),causes immunosuppression in young chicken by destroying the precursors of B lymphocytes in the bursal of Fabricius. In 1957, the disease first broke out in broilers in Gumboro, Telahua state, America. In the 1980s, the variation strain of IBDV was first appeared in America. Acute IBD caused by very virulent infectious bursal disease virus (vvIBDV) was first reported in Europe lately 1980s. The acute disease was then transmitted into China in the early 1990s, and rapidly spreaded all over Asia and other major parts of the world. The IBDV had caused the significant economic losses in poultry industry for a long time. Through research, people have confirmed that VP2 play a critical role at the induction of NA, mutation of antigen, apoptosis of cell et al., However, there is no reports about receptor protein of IBDV up to now.The development of the yeast two-hybrid system provided a genetic means to identify proteins that physically interact in vivo.The goal of the system is to isolate a protein or proteins that interact with a "bait" protein, this bait protein can be almost any protein from any organism. Over the past ten years, this system and its variants have been extremely useful for detecting and analyzing protein-protein interactions in many fields. One purpose of the research is screening the interactive coding gene with IBDV Gt VP2 from CEF cDNA library by the yeast two-hybrid system. Addition, we take a preliminary research for discoverable gene, all those can as a substantial foundation for us to research pathogenic mechanism of IBDV and prevention of IBD.The following are the main contents of this reseach:1. Construction of chicken embryo fibroblasts cDNA expression library by gateway technology. The mRNA was extracted from chicken embryonic fibroblast. Moreover, single-strand cDNA and double-strand cDNA were synthesized by using biotin-conjugated Oligo (dT) primer in turn. The double-strand cDNA was ligated Adapter and then purified by the cDNA Size Fractionation Columns. After BP recombination reaction, an cDNA entry library was constructed with a titer of 1×10⁶cfu/mL, total clones of 1.2×107 cfu and an average insertion size of about 2243bp. After LR recombination reaction, the cDNA entry library was transformed into expression library which took on a titer of 5×105cfu/mL, total clones of 5.5×10⁶ cfu and an average insertion size of about 2411bp. The results indicate that the constructed cDNA expression library performs a remarkable high value in both recombination rate and library coverage. As a result, the cDNA expression library, with its good quality, may facilitate to identify the receptors associated with the resistance against IBDV in chicken embryonic fibroblast and to cast new light on the mechanism of cellular tropism. Moreover, it may also provide data of chicken embryonic fibroblast in transcription level and may be helpful to study its biological functions.2. Through BP and LR reaction, the fragments of infectious bursal disease virus (IBDV) Gt VP2 was cloned into pDEST-32 vector by Gateway technology, after confirmation with restriction enzyme digestion, PCR and sequence analysis, the plasmids were transformed into the yeast cell MaV203 with ployethylene glycol/lithiuma acetate method, then the growths of transformation were observed on the SC-Trp-Leu-His selective plate, to detect the self-activation of bait vectors. At last, determination 3AT density and verification the bait vector neither the ability of outonomous reporter gene activation, nor toxicity to the yeast host-cells.3. Transform bait plasmid into yeast competent cell, then to select the positive clone as the yeast competent cell for CEF library plasmid. Through X-gal test and three selective medium, we found 19 postive clones, extraction the plasmid and sequence. In the result, we obtain two gene segments (similar Gallus gallus gene as 99.9%), they are 800bp and 350bp respectively. Continue, construction prey plasmid with two gene segments, to test the interaction between two segments with Gt VP2 again.4. Delete the termination codon by PCR technology, then little segment (CR2₁₁) was cloned into expressing vector pET32a and vector pCAGGS, the recombinants were transformed into E.coli DH5α. The recombinant plasmid was identified by restriction enzyme digestion, PCR and sequence analysis and named pET32a-CR2₁₁ and pCAGGS-CR2₁₁. The recombinant plasmid (pET32a-CR2₁₁) was transformed into E.coli BL21(DE3) and induced by IPTG. SDS-PAGE results indicated that the fusion protein was about 31kDa. The pCAGGS -CR2₁₁ was transfected into PK15 cell, IFA showed that there are strong green fluorescence on the cell hole (PK15), It proved the interactive role between Gt VP2 and CR2₁₁ in vivo.