Identification and verification of α-11 giardin-interacting protein / 中国血吸虫病防治杂志

Objective To identify and verify the interacting protein of α-11 giardin, so as provide the experimental evidence for studies on the α-11 giardin function. Methods The yeast two-hybrid cDNA library of the Giardia lambia C2 strain and the bait plasmid of α-11 giardin were constructed. All proteins interacting with α-11 giardin were screened using the yeast two-hybrid system. α-11 giardin and all screened potential interacting protein genes were constructed into pBiFc-Vc-155 and pBiFc-Vn-173 plasmids, and co-transfected into the breast cancer cell line MDA-MB-231. The interactions between α-11 giardin and interacting proteins were verified using bimolecular fluorescence complementation (BiFC). Results The yeast two-hybrid G. lambia cDNA library which was quantified at 2.715 × 107 colony-forming units (CFU) and the bait plasmid containing α-11 giardin gene without an autoactivation activity were constructed. Following two-round positive screening with the yeast two-hybrid system, two potential proteins interacting with α-11 giardin were screened, including eukaryotic translation initiation factor 5A (EIF5A), calmodulin-dependent protein kinase (CAMKL) and nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH), hypothetical protein 1 (GL50803_95880), hypothetical protein 2 (GL50803_87261) and a protein from Giardia canis virus. The α-11 giardin and EIF5A genes were transfected into the pBiFc-Vc-155 and pBiFc-Vn-173 plasmids using BiFC, and the recombinant plasmids pBiFc-Vc-155-α-11 and pBiFc-Vn-173-EIF5A were co-tranfected into MDA-MB-231 cells, which displayed green fluorescence under a microscope, indicating the interaction between α-11 giardin and EIF5A protein in cells. Conclusion The yeast two-hybrid cDNA library of the G. lambia C2 strain has been successfully constructed, and six potential protein interacting with α-11 giardin have been identified, including EIF5A that interacts with α-11 giardin in cells.

Screening CVB3 VP3 interacting proteins from cDNA library of human heart by yeast two hybridization / 中国人兽共患病学报

To screen interaction proteins of CVB3 VP3 from cDNA library of human heart ,yeast two hybridization was conducted in this study .The bait plasmid pGBKT7-VP3 was constructed ,VP3 fusion protein and its self-activation in AH109 yeast cells was then detected .The positive clones were confirmed by PCR amplification of cDNA inserts ,Alu I digesting ,DNA sequencing ,and Blasting were used to sort positive colonies to eliminate duplicates .Positive clones were confirmed by one-to-one yeast two hybridization ,and them were sequenced and analyzed for homology .Theα-galactosidase assay was performed to detect the interaction strength .Totally ,10 positive proteins interacting with VP3 of CVB3 were obtained by homology analy-sis,namely,EIF4A2,HADHB,GAPDH,ASPG,ACTA1,TNNI3,CKM,LMOD3,ERGIC1,and ALDH2.The strength of interactions between VP3 and 10 candidate proteins were proved byα-galactosidase assay .This study will contribute to explore the CVB3 VP3 function on molecular level and provides some new clues to explain the pathogenic mechanism of myo-carditis and cardiomyopathy .

Interaction between human cytomegalovirus UL136 protein and ATP1B1 protein

Interplay between the host and human cytomegalovirus (HCMV) has a pivotal role in the outcome of infection. A region (referred to as UL/b’) present in the Toledo strain of HCMV and low passage clinical isolates contains 19 additional genes, which are absent in the highly passaged laboratory strain AD169. Products of the UL/b’ genes may determine the manifestations of HCMV infection in vivo. However, little is known about the host factors, which interact with UL/b’ proteins. This study was conducted to investigate the function of the HCMV UL136 protein. By yeast two-hybrid screening, the β1 subunit of the host Na+/K+-ATPase (ATP1B1) was identified to be a candidate protein, which interacts with the HCMV UL136 protein. The interaction was further evaluated both in vitro by pull-down assay and in vivo by immunofluorescent co-localization. The results showed that the UL136 protein can interact with ATP1B1 in vitro. Co-localization of UL136-EGFP and ATP1B1-DsRed in cell membranes suggests that ATP1B1 was a partner of the UL136 protein. It can be proposed that the HCMV UL136 protein may have important roles in processes such as cell-to-cell spread, and in maintaining cell osmotic pressure and intracellular ion homeostasis during HCMV infection.