Construction Of A Yeast Two-hybrid CDNA Library For Apis Cerana Larvae And Screening Its Interaction Protein With VP3 Of Sacbrood Virus

ObjectiveTo screen and characterize interaction protein of Chinese sacbrood bee virus(CSBV)VP3 from its host Apis cerana larvae based on the cDNA library construction technology and yeast two-hybrid system.The identified interactions of the proteins would support for the further study of the CSBV pathogenesis mechanism.MethodsTotal RNA was extracted from the Apis cerana larvae then the synthesized cDNA fragment was ligated with p GADT7 vector to construct yeast cDNA library of Apis cerana larvae.The titer of the yielding library and the size of the inserted cDNA fragment were examined and verified.The CSBV VP3 gene was subcloned into eukaryotic expression vector pGBKT7 to construct the bait palsmid pGBKT7-VP3,and then the bait plasmid pGBKT7-VP3 was transferred into yeast competent cells for the subsequent detection of its self-activating ability as well the toxic effect on yeast cells.The yeast cells harboring the bait plasmid pGBKT7-VP3 were fused with the cDNA library of Apis cerana larvae.The positive clones selected by yeast two-hybrid system were picked as PCR templates,and the subsequent amplified PCR products were sequenced.The sequencing results of the capture protein were analyzed with reference to the relevant literature,then VP3 was predicted to interact with possible Galectin1.The obtained Galectin1 gene was cloned into p GADT7 vector and subjected to yeast return assay.The recombinant prokaryotic plasmid p GEX6P-1-Galectin1 and p ET28-a-vp3 were constructed and expressed through optimization of the codon bias of Escherichia coli.Interaction of expressed Galectin1 with VP3 protein was verified by Far-western blot technology.ResultsThe extracted highly purified non-degradable RNA meets the requirements for the construction of cDNA library of Apis cerana larvae.The capacity of cDNA library was 1.25 � 107 cfu and the cell titer was 2.5 � 106 cfu/ m L,Twenty-four positive clones were randomly selected to amplify and operate agarose gel electrophoresis,the results showed that the inserted fragments were evenly distributed and its size was 1.5-3 kbp,there was no blank in 24 positive clones electrophoresis lanes,which suggested that the recombination rate of the library was 100%.The constructed bait plasmid pGBKT7-VP3 was transferred into yeast competent cells had no self-activating ability and non-toxic to host cells.The host protein Galectin1,which interacts with CSBV VP3 protein,was then screened from the cDNA library of Apis cerana larvae.The recombinant proteins from p GEX6P-1-Galectin and p ET-28a-2 were obtained by codon optimization.VP3 was highly expressed in Escherichia coli and the recombinant proteins were purified respectively.The Galectin1 protein interacting with the VP3 protein was confirmed preliminary by yeast return test and Far-western blot assay.Conclusions1.The cDNA library of Apis cerana larvae was successfully constructed.2.The pGBKT7-VP3 bait plasmid was successfully constructed and its interaction protein Galectin1 was screened out by the yeast two-hybrid system.3.The interactions of galectin1 and VP3 was confirmed by Yeast return test and Far-western blot assay.