Establishment And Application Of Giardia Duodenalis Fluorescence Labeling Method And PCR Detection Method Based On Giardin ?-12

Giardia duodenalis is a zoonotic parasite that causes host diarrhea.Giardisis caused by Giardia duodenalis outbreaks in many places.The symptoms of the disease are diarrhea,dehydration,abdominal pain,nausea,vomiting,weight loss and so on.Nearly 280 million people around the world are infected every year.In addition to infecting humans,it also infects livestock(cattle,sheep,pigs,rabbits,etc.),wild animals(foxes,wolves,kangaroos,camels,etc.),and pets(cats,dogs,chinchillas,etc.).The epidemic of the disease will cause serious public health problems and economic losses to animal husbandry.Therefore,the rapid and accurate diagnosis of Giardia duodenalis is one of great significances to the health and safety of animal husbandry.Giardia duodenalis is an anaerobic protozoan and localizes the mucosal epithelium of the small intestine of the host.It not only has the characteristics of eukaryotes and prokaryotes,but also has its own biological characteristics,so Giardia duodenalis is a meaningful research object of cell biology and biological evolution.Fluorescence labeling method can be used in the biological study of Giardia duodenalis to visualize the related activities at the genetic level.The commonly used Giardia duodenalis labeling fluorescein GFP and SNAP tags have some limitations in the application of Giardia duodenalis.Sortase A is a new transpeptidase which can be used for protein modification and labeling in living cells and in vitro.it is widely used in the field of biotechnology.Previous studies in our laboratory have found that giardin?-12 has a high specificity and high expression which has been applied to the establishment of indirect ELISA detection method.Therefore,this experiment intended to establish a Giardia duodenalis fluorescence labeling method and PCR detection method based on giardin?-12.The main research contents are as follows:Based on the establishment of Giardia duodenalis labeling method of giardin?-12,firstly,giardin?-12 was ligated with fluorescent dye in vitro.SortaseA protein was expressed,purified,concentrated and its activity was detected.The expression vector of pET-28-giardin-?-12-LEPTG was constructed,and the giardin-?-12-LEPTG protein was obtained after expression and purification,and the molecular weight was about 36kDa.The obtained SortaseA,giardin-?-12-LEPTG and polyG(GGG)-GFP(about 26kDa)were co-incubated and identified by SDS-PAGE analysis.The giardin-?-12-LEPTGGG-GFP binding protein with molecular weight of about 62kDa was successfully obtained.The specific binding of giardin?-12 with fluorescent dye in vitro was successfully realized.The experiment of fluorescence labeling method in Giardia duodenalis:the viral vector pC631-giardin-?-12 was successfully constructed and transferred into Giardia duodenalis by transcription and electroporation in vitro,and the Giardia duodenalis strain with hygromycin resistance was obtained.The morphological structure of the target strain was well-formed and the growth curve was not changed under scanning electron microscope,and the target strain was successfully obtained and can be stablely passaged for more than 20 generations.The strain which could stably express giardin-?-12-LEPTG was successfully obtained after the detection by Real-time PCR.The target strain was co-incubated with SortaseA and polyG-FITC,and Giardia duodenalis with fluorescence labeling was successfully observed under laser confocal.The labeling method also successfully confirmed the cellular localization of giardia?-12 in Giardia duodenalis.The establishment of PCR detection method of Giardia duodenalis based on giardin?-12:according to the sequence of giardin?-12 gene in NCBI,the highly conserved gene fragment was selected to design and synthesize primers,and the reaction conditions were optimized.The annealing temperature and extension time of the PCR method were selected to verify the specificity and sensitivity of the method.The results showed that the best annealing temperature was 62?,extension time was30s,and the specificity was well.The sensitivity test results showed that a minimum of 2×10~1 Giardia duodenalis cysts per gram of sample could be detected.The established Giardia duodenalis PCR detection method based on giardin?-12 was used to detect 283 sheep fecal samples from farms around Changchun.The positive rate of Giardia duodenalis was 13.7%.The genotype of Giardia duodenalis was assembling A.In summary,the fluorescence labeling method and PCR detection method of Giardia duodenalis were successfully established in this study,which provided experimental ideas and technical means for understanding more biological functions of Giardia duodenalis,and provided a basis for further diagnosis,prevention,and control of Giardiasis.