Screening Proteins To Interact With M1 By Yeast Two-hybrid System

The Ml protein of avian influenza (AI) viruses is a multifunctional protein with important roles in the life cycles of the viruses. The theoretic molecular of the Ml protein mass is 28kDa. The thesis are divided into two part, in the first part, according to the sequence of Ml gene published in NCBI, primers were designed and the cDNA fragment was amplified by PCR with the template of the pGEM-T-M1 (presented by Prof.Liu Jin-hua in Chinese Agriculture University, belonged to H9N2), sequenced and compared with other sequence (A/chicken/Heilongjiang/48/01(H9N2)), the identity exceeds 99%. The result indicated that the cDNA fragment could be used as Ml gene belonged to H9N2. The yeast two-hybrid system was used to screen proteins to interact with Ml, we got five proteins which names were human C1q, CAP, AHA1, Adolase B, PPIE. And the interactions between the Ml protein and Clq, CAP, AHA1, Adolase B, PPIE were confirmed by the yeast two-hybrid system.In the second part, GST-pull down assay was used to confirme the interaction between the Ml protein and AHA1, PPIE, PPIA. According to the sequences of the AHA1, PPIE, PPIA genes published in NCBI, three primers were designed and these cDNA fragments were amplified by PCR with the template of the Kidney MATCHMAKER cDNA library, sequenced and compared With sequences published in NCBI, the identities were 100%. Then, AHA1, PPIE, PPIA were expressed in the prokaryotic cells. GST-pull down assays were used to confirme the interaction between the Ml protein and AHA1, PPIE, PPIA. All of the results implicated that the Ml protein may mediate the interaction between protein and protein, which functions in assembly and budding of influenza virus.