Isolation And Culture Of Primary Tupaia Intestinal Epithelial Cells And The Infection Of RV On It

Rotavirus(RV) is the major pathogen of diarrhea disease in infants, whichresponsible for nearly 800,000 deaths per year worldwide. There is no effectivemedicine to treat rotavirus infection by now,and the protective efficiency of existingvaccine are very different in varied areas. In addition, the existing vaccine are expensive and having the potential safety problems,it is urgen matter to develop a new vaccine which is safer,cheaper and more efficient. The reliable animal model is the guaranteeof vaccine development, and helps understand the process of virus infection and pathogenesis.Rotavirus infection animal models including gnotobiotic pigs, monkeys, mice and so on.Because of the limitations of kinship, experiment duration, anatomical characteristics,the studyofRV infection and causative pathogenesismechanismsare subject to restrictions.However,in recent years, a lot of studies reported thatChinese Tree shrew(Tupaia belangeri) have many valuable features which indicate tree shrew potential to be utilize as experimental animal for RV research like closly related with human as compared with mouse, small body mass,short life period, low production cast and faster growth rate.The isolated and culture of the tree shrew primary intestinal epithelial cells is the key toestablish tree shrewsinfection model in vitro and applied basic research.In this study, we establish a rotavirus TaqMan fluorescent quantitative PCR detection method. Based on the VP6 gene, a set of primerand TaqMan probe were designed.The VP6 gene fragmentwasamplifiedand cloned into the PCDNA3.1+ vector. RNA was transcribed in vitroand serial diluted to establish the externalstandards.With the optimization for PCRparameters,the optimized primer concentration was determined as 250nM and probe concentration as 300nM. The human coxsackievirus and reoviruscouldn’t be detectedwhich provedthe high specificity of this method. The low limitation of this method was detected to be less than 5copies/μL, and variation coefficientless than 1%.Finally, a TaqMan real-time fluorescence quantitative PCR for human rotavirusdetection was established,which may providea new approachfor the RV diagnosis, food safety inspection and epidemiological investigation.Three methods were adopted in this experiment, including Pancreatic enzyme, collagen enzyme and a mixture of collagenase type (xI) and neutral protease (type Ⅰ), the method of collagenase type (Ⅺ) and neutral protease (type Ⅰ) for obtaining small intestinal epithelial cellsismorestable.Thepuretreeshrewsprimarysmallintestinalepithelialcellswereobtainedusingd igestionliminationmethodandidentifiedbyKeratin18antibody.Theresultispositiveandshowsthat obtainedcellsaretreeshrewsprimarysmallintestinalepithelialcells.It prove that tree shrews primary intestinal epithelial cells can be infected with RV by using nestedpolymerase chain reaction (PCR),quantitative PCR,western blot,and IF,Serial dilution method and fluorescence quantitative PCR were used to detect rotavirus optimal MOI and proliferative properties in intestine epithelial cells.The viral load was biggest when tree shrew primary intestinal epithelial cells were infected with human rotavirus at MOI of three. Dilution and fluorescence quantitative PCR were used to detect the viral titer and load changes of rotavirus proliferation. The virus growth curves were drawn. The viral titer and load was low at the first day, had an exponential growth at 2-3 days, reached a plateau at 3 days andshoweda trend of decreasewith the extension of time. Despite the low level of infection, the results show that tree shrew can regard as RV infection animal model for subsequent research.In summary, this study successfully constructed the tree shrews infection model in vitro,Themodel for the development of rotavirus vaccines, assessment of the effectiveness on antibody treatment and the study based on the mechanism of viral infection has important significance.