Construction And Identification Of Pseudorabies Virus MicroRNA Library

Porcine pseudorabies(PR) is caused by pseudorabies virus(PRV), which isextremely contagious and acute infectious disease. PRV belongs to Alphaherpesvirinaesubfamily that causes huge economic loss in pig industry all over the world. Recently,studies have shown that the herpes virus-encoded miRNAs play a vital role in the viralreplication and regulation. MicroRNAs(miRNAs) have approximately22nucleotidesthat are a class of non-coding small RNA distribute extensively in human genome andother organisms genome. They regulate gene expression through sequence specificinteractions with target mRNAs resulting in translational repression or mRNAdegradation. the research of PRV-encoded miRNAs is still very limited. Therefore, thestudy of PRV miRNA has considerable academic value.In this study, PRV was cultured in PK-15cells for viral proliferation, thencollected PRV-infected cells in different time periods after inoculation. After extractingthe total RNA, denatured polyacrylamide gel was used to enrich18-26nt RNA. Bothends of the small RNA molecules was ligated with two different bio-adapters. RT-PCR,connecting the target DNA fragments and plasmid vectors to transform theengineereing d bacteria. Finally, the construction of pseudorabies virus microRNAlibrary was completed. Randomly picked some clones from the positive clones, PCRamplification to identify the characteristics of the clones and sequenced, we got20clones of the correct size and used bioinformatics method for preliminary screening ofmiRNA. The experiment results show that the cDNA library of PRV miRNA wassuccessfully constructed through the enrichment of small RNA and small RNA cloning.Combined sequence analysis and PRV bioinformatics screening, except for self-connected adapters or coding RNA degradation fragments, we got a pig miRNA and aPRV miRNA, which confirmed the formation and existence of miRNA in proliferationprocess of PRV that cultured in vitro. The cDNA library of pseudorabies virus microRNA was constructed successfully,which laid the foundation for the research of pseudorabies virus miRNA. But cloningmagnitude of the cDNA library is very limited, therefore, this experiment involved thepseudorabies virus microRNAs is just a beginning.