Expression And Construction Of Eukaryotic Expression Vector Of Two Crypto Sporidium Parvum Mirna

Cryptosporidiosis that caused by Cryptosporidium was a worldwide distribution and opportunistic zoonotic parasitic diseases. The main clinical symptoms is self-limited watery diarrhea. Nowadays, there is no effective treatment drug and vaccine for this disease. At the same time, there also is not the reports about the discovery and study of the Crypotosporidium microRNA (miRNA). The research of miRNA not only help researcher to understand the growth and development mechanism of Cryptosporidium, but also contributes to discovery new drug targets and to design new vaccines.1. The microRNA cp-miR2980and cp-miRm0001sequence information were obtained through the laboratory work of construction of cryptosporidium parvum small RNA library in early days including the sequencing and bioinformatics analysis of cryptosporidium parvum. The total RNA of different kind of cryptosporidium was extracted by using Trizol reagent and liquid nitrogen, then to use the kit for separation and enrichment of small RNA, poly(A) polymerase treatment, reusing the small reverse transcription primers and reverse transcription kit for reverse transcription reaction. The expression of cp-miR2980and cp-miR0001was detected by using real-time PCR in C. andersoni, C. parvum, C. baileyi, Cryptosporidium mouse genotype, C. cuniculus, C. suis6species of cryptosporidium oocysts. The Real-time PCR amplification products were identified by using agarose gel electrophoresis. The results show that:the level of expression of cp-miR2980and cp-miR0001were different in different genotypes of cryptosporidium parvum oocysts. At the same time, the PCR positive strip was detected by using agrose gel electrophoresis, which confirms that the expression of cp-miR2980and cp-miR0001were different in different genotypes of cryptosporidium oocysts. It is further confirmed that the Real-time PCR was a efficient, rapid, sensitive, good specificity, low cost method to detect the expression level of miRNA.2. The cp-miR2980precursor and cp-miR0001precursor were amplified from the cryptosporidium parvum genomic DNA. Then the cp-miR2980precursor and cp-miR0001precursor were cloned into pMD18-T vector. The amplified precursor was then subcloned into pVAX I vector and identified with restriction endonuclease digestion and sequencing. The recombinant plasmid pVAX-cp-miR2980and pVAX-cp-miR0001were respectively transfected into HCT-8cells. Total RNA was extracted and the expression of cp-miR2980and cp-miRm0001were detected by RT-PCR. The results show that:the pVAX-cp-miR2980and pVAX-cp-miRm0001were successfully constructed and the pVAX-cp-miR2980and pVAX-cp-miRm0001can respectively express cp-miR2980and cp-rmiRm0001in HCT-8cells. This results were helpful to further research the biological function of cp-miR2980and cp-miRm0001.3. The information of Cryptosporidium parvum Iowa Ⅱ RAD50gene was predicted through the liborary work of construction of Cryptosporidium parvum small RNA library sequencing and bioinformatic analysis. The3’UTR fragment of Cryptosporidium parvum Iowa ⅡRAD50gene was amplified from Cryptosporidium parvum oocysts genomic DNA and cloned into pMD18-T vector. The amplified3’ UTR fragment of Cryptosporidium parvum Iowa ⅡRAD50was then subcloned into PGL3vector and identified with restriction endonuclease digestion and sequencing. The luciferase reporter vector and pVAX-cp-pre-miRm0001were transfected into HCT-8cells and the pRL-SV40plasmid was co-transfected as the internal control. The results show that:the3’UTR fragment of Cryptosporidium parvum Iowa Ⅱ RAD50gene was successfully cloned into the pGL3reporter vector, which authenticated by restriction endonuclease digestion and DNA sequencing. The luciferase activity of the experimental group was significantly decreased by comparing to the control group, which indicate that the cp-miRm0001has the inhibitory effect to the luciferase reporter vector of the3’UTR fragment of Cryptosporidium parvum Iowa ⅡRAD50gene. This inhibitory effect was due to the interaction of cp-miRm0001and the3’UTR fragment of Cryptosporidium parvum Iowa Ⅱ RAD50gene. The study of construction of luciferase reporter gene vector provides the foundation for to further study of the mechanism of microRNA regulation of target gene expression and the function of Cryptosporidium parvum microRNA.