Construction And Screening Of A Full-Length CDNA Library Of Infant's Small Intestine By RRS System

Background Rotavirus (RV) is the main pathogen of infant's diarrhea, accounting for as many as five million deaths annually. Because the morbility and infection rate are high either in developed or in developing country, it is more necessary to develop an effective vaccine and therapeutic strategies to combat virus. But the premise is we need a fundamental understanding of the molecular mechanism between virus and the host cells.The first and necessary step for RV infection is its binding to the receptors on the cell membrane. Up to now, the natures of those receptors are unclear because few quantities on the host cell, complex natures as well as technique's limitation. Full-length cDNA is not only helpful in appraising the extron - intron boundary in its genome team sequence, analyzing gene code area and understanding gene's copying and translation function, but also can be used to analyze the human genome expression profile, function and structure.The yeast two-hybrid system has been one of the important methods for research of interaction between proteins since it was founded. Traditional two-hybrid locates the interaction in the nucleus, while the Ras Recruitment System (RRS) developed in recent years can locate which on the cell membrane and has more advantages such as high sensitivity and less false positive. The principle of the RRS is as follow: In the yeast temperature-sensitive cdc25-2 strain, the yeast Ras is rendered inactive at the restrictive temperature (36℃) due to lack of a functional cdc25-2 guanyl nucleotide exchange factor. But if we introduce a normal GEF (Ras protein) which could be localized on membrane through protein-protein interaction, the yeast temperature sensitive mutant strain cdc25-2 can grow at 36℃because of Ras signaling pathway activation.Objectives In our study, we constructed a full-length cDNA library of infant's small intestine tissue and then cloned it to the plasmid pUra-MΔpolyA to obtain a prey plasmid suitable for RRS. To understand the molecular mechanism of interactions between RV and host cell, we cotransformed bait plasmid (pMet425-Myc-Ras-VP4) and prey plasmid (pUra-MΔpolyA-cDNA) into the yeast temperature sensitive mutant strain cdc25-2 to screen the proteins interacted with RV VP4 by RRS.Methods1. Total RNA from small intestine tissue was extracted by Trizol. After mRNA purified, cDNA was synthesized by reverse transcription, and cDNA fragments larger than 500bp were ligated with dephosphorylated arms of pUra-MΔpolyA vectors. The recombination vectors were transformed into DH5αby CaCl2. Calculating the library capacity and identifing the size of insert fragment by digesting with restriction enzymes EcoRI/XhoI.2. Bait plasmid (pMet425-Myc-Ras-VP4) and prey plasmid (pUra-MΔpolyA-cDNA) were cotransformed into the yeast temperature sensitive mutant strain cdc25-2. Screening the proteins interacted with RV VP4 by changing the components of the midum and environmental temperature to get rid of potential false positive.Results1. The full-length cDNA library contained 6×107 recombine clones. The insert size ranged from 0.5 to 2 kb, average 1.5kb.2. Screening positive clones by RRS, and detecting the sequences.Conclusions1. A high quality full-length cDNA library from infant's small intestine tissue was first constructed successfully which can be used for screening by RRS.2. The possible protein receptor from cDNA library of infant's small intestine tissue were found out which interacted with RV coat protein VP4 by RRS, waiting for the results of sequencing.