Acquired Of The Full-length CDNA Of HCMV UL148 Gene And Its Bioinformatical Analysis

BackgroundHuman cytomegalovirus (HCMV) is ubiquitously distributed in the human population. Usually, HCMV asymptomatically infects the host and establishes life-long latency. In immunologically immature and immunocompromised individuals, virus reactivation from latency can result in serious and life-threatening disease. Results of this study demonstrate that the unique long (UL/ b') region of HCMV genome, which encodes protein highly involved in viral pathogenicity.This boundary includes 19 ORF (UL133-UL151), however, systematic analysis of the transcripts for this region is still insufficient.our previous study preliminarily first confirmed that UL148 et al five gene exist the mRNA expression level by RT-PCR, but the transcripts of all the genetic information is the unclear. In this study we Acquired the transcripts situation of UL148 gene by rapid amplification of cDNA end (RACE) technology , analyzed the transcripts information, researched the possible function of UL148 gene.Objective1. To obtain the full lengh cDNA of HCMV UL148 gene transcripts by rapid amplification of cDNA ends (RACE) technology in HCMV.2. Using bioinformatics methods to analyse the transcripts information of UL148 gene.3. Preliminary forecast the function of UL148 encoding protein, setting up foundation for further research of UL148 gene.MethodsInoculate HCMV to well growth HFF cell;observe cytopathic effect(CPE) by microscope; after the HFF cell appear cytopathic effect, extraction viral DNA from supernatant fluid;according to the conservative gene IE and LA. desigening two pair primer sequence ,and amplified the HCMV conservative genes at the same time by multiple PCR to identify the virus;collect cytopathic cell, extraction viral RNA; according to the foregone segmental cDNA sequence, respectively design specificity primers in 5' and 3' termini,corresponding to 5'RACE and 3'RACE Adaptor primers, obtained 5' termini and 3' termini of cDNA of HCMV UL148 by Rapid Amplification of cDNA Ends technology.5'and 3'RACE products were cloned into pMD18-T vector and their sequences were confirmed by sequencing subsequently; The UL148 genes primers were designed according to the result of RACE. Amplified full lengh cDNA of HCMV UL148 gene by RT-PCR. the result were analyzed in GenBank. Use bioinformatics methods to analyse UL148 protein's properties and characteristics, preliminary predict UL148 gene's biological function.ResultsHFF cell were successfully infected by HCMV, the cell were present typical cytopathic effect,. Using multiple PCR, detect virus replication must gene: IE and LA genes to identify virus, the results were agree with the expected size bands.extraction viral RNA. Using RACE assay, we obtained and cloned the 5 'and 3' termini of UL148 gene, sequencing results show that its 5'termini region long 496bp, 3'termini region long 995bp; The full length cDNA of UL148 gene is 2442 bp, that was amplified by RT-PCR technology, 3'-coterminal of transcripts in the UL148 and UL132 region including 11 poly A tail, were located approximately 20 bp downstream from each potential poly A signal. Bioinformatics method analysis shows that UL148 gene transcription product coding protein relative molecular weight for 36.473kD, theoretical pI for 9.15, for a alkali protein, Formula is C1664H2585N453O449S11,no introns sequence, open reading code box contains 316 amino acids. UL148 genetic encoding protein 286 ~ 308 amino acids are transmembrane site, speculated that it was a transmembrane protein.Conclusion1. Successfully obtained the full length cDNA of HCMV UL148 and UL132 gene by Rapid Amplification of cDNA Ends technology. The cDNA sequence is 2442bp in size. 2. The results indicated UL148 gene and UL132 gene is a transcrips, there is a 3'-coterminal.3. The bioinformatics analysed indicated UL148 encoding protein is a transmembrane protein.4. Extrapolated that UL148 gene possibly has certain function in the HCMV pathogenesis aspect.