Construction Of A Membrane Protein Yeast Two-hybrid Library For Apis Cerana Larvae And Screening Host Protein Interacted With Chinese Sacbrood Virus VP2

ObjectiveThe Apis cerana larvae a membrane protein cDNA was ligated to pPR3-N vector to construct the Chinese larvae membrane protein yeast cDNA library using FullCoV technique,use this library,the host protein interacted with Chinese sacbrood bee virus (CSBV)structural protein VP2 was screened,Which lays the foundation for further study on function of the CSBV VP2.MethodsThe total RNA was extracted from the 2~3 day-old larvae,after mRNA was isolated,cDNA of the first strand was synthesized under the action of reverse transcriptase and double-stranded cDNA was synthesized.After the linker with the recombination sequence was added to the 5'end of the double-stranded cDNA,it was ligated with the vector pPR3-N by FullCoV technology,and then the ligation product was electro transformed into competent cells(DH10B)to construct the larval membrane protein yeast cDNA library,the titer of the yielding library and the size of the inserted cDNA fragment were examined and verified.The CSBV VP2 gene was cloned into vector pBT3STE?pBT3SUC respectively to construct the bait palsmid pBT3STE-VP2 and pBT3SUC-VP2,and then the bait plasmid pBT3STE-VP2and pBT3SUC-VP2 was transferred into yeast competent cells NMY32,and its function and self-activating ability in yeast cells was detected.On this basic,the yeast cells harboring the bait plasmid were fused with 25 mg cDNA library for Apis cerana larve palsmid,scribble to SD-TLH defect plates and incubate at 30?for 72 h.All colonies were then rinsed with PBS solutionandtransferredtoSD-TLH-deficientmediumcontaining3mM3-amino-1,2,4-triazole(3AT)and incubated 72 h,followed by single colony was diluted and cultured respectively on SD-TL and SD-TLHA-deficient medium which contained 3 mM,10mM,40 mM and 60 mM 3AT.After culturing at 30?for 24 h,the colony growth was observed.Then,the positive clones which were initially determined to be positive were picked,and the colony was detected by PCR using pPR3N detection primer.The yeast plasmids were extracted from the yeast colonies that amplified to specific fragments,and transformed into competent cells(DH5?).Plasmids were extracted and subjected to PCR again.The plasmids that has same PCR bands both two PCR were sequenced.Finally,we compared the sequencing results by BLAST to speculate the host proteins that may interact with CSBV VP2.ResultsTo successfully construction Apis cerana larvae a membrane protein yeast two-hybrid cDNA library by the FullCoV technique.The capacity of cDNA library was 1.5�10~7 cfu and the cell titer was 3�10~6 cfu/mL,the recombination rate of the library was 100%.The constructed bait plasmid pBT3STE-VP2,pBT3SUC-VP2 function and self-activation test results showed that both bait plasmids can be expressed in yeast cells NMY32 and no self-activation ability,the result of bait plasmid pBT3STE-VP2 was better than pBT3SUC-VP2,so it will be used in subsequent experiments.The interaction between Apis cerana larvae a membrane protein yeast two-hybrid cDNA library and yeast cells containing bait plasmid pBT3STE-VP2 showed that 58 positive clones were initially screened on SD-TLHA deficient medium with 3AT concentration of 60mM,the results of bacterial PCR showed that 35 of them were amplified 600-2500 bp of bands,the plasmids of these 35positive clones were transformed into DH5?amplified and then detected by PCR,A total of25 plasmids with same PCR bands were obtained and sequenced.Sequencing results were compared with the sequences in GenBank database for BLAST analysis,the results showed that there may be 12 kinds of host proteins that may interact with VP2 protein of CSBV,include Apis cerana 40S ribosomal protein,Apis cerana 60S ribosomal protein,Apis cerana heat shock 70 kDa protein,Apis cerana cerana mitochondrial DNA,Apis cerana probable salivary secreted peptide,Apis cerana translocon-associated protein,Apis cerana ATP synthase lipid-binding protein,Apis cerana ankyrin repeat domain-containing protein,Apis cerana serine/arginine-rich splicing factor,Apis cerana U6 snRNA-associated Sm-like protein,Apis cerana coiledl-helix containing protein,Apis cerana uncharacterized protein.Conclusions1.Apis cerana larvae a membrane protein yeast two-hybrid cDNA library successfully constructioned by the FullCoV technique.2.The pBT3STE-VP2 and pBT3SUC-VP2 bait plasmids were constructed,the bait plasmids had no self-activation ability and good expression in yeast cells,the expression level of pBT3STE-VP2 was better than that of pBT3SUC-VP2.3.A total of 12 host proteins capable of interacting with the VP2 protein of CSBV were screened through the interaction between the interaction between Apis cerana larvae a membrane protein yeast two-hybrid cDNA library and yeast cells containing bait plasmid pBT3STE-VP2.