Construction Of Human Cytomegalovirus CDNA Expressing Library And Screening Of Pp65 Positive Clones

Objective: Construct HCMV AD 169 strain and isolated strain genome cDNA expressing library, which provide a sound basis for further studying of the structural character and function of their genome Methods: HF cell which were infected by 1-ICMV AD169 sftain(MON1O) and HCMV isolated strain(MOI=1O)cells were collected at 96h p.i, mRNA contained poly(A) was isolated by chronimatography on oligo(dT) cellulose, whose ratio of 0D260/0D280 was 2.1, then reverse transcripted into eDNA, second- strand DNA synthesis was done by using Rnase H and DNA polymerase 1, and E. co/i ligase to replace the RNA strand with deoxynucleotides . After methylation and EcoR I linker addition the cDNA was cloned into EcoR I-digested lambda gtl 1 .The ligated DNA was packaged , then transinfected host cells ,ie LE392 and Yl 090.The libray was screened by IPTG inducing and X-gal colouring , immune blot with anti-HCMV mouse convalescent sera , nucleic acid hybridization with DIG- labled HCMV pp65 gene probe and confirmed by PCR. Results: HCMV eDNA expressing libraries were constructed , their volume were 3.6 X lO6pfulml and 3.3 X 1 O6pfuIml respectively,recombinatant efficiency of AD 169 was up to 76%, 168 positive clones of ADI 69 were tested by immune blot, 34 positive clones were obtained by dot nucleic acid hybridization with pp65 probe, 3 positive clones were amplified by HCMV pp65 all length primer. Conclusion:HCMV AD169 strain and isolated strain cDNA expressing library have been constructed and 3 pp65 positive clones frome AD 169 cDNA expressing library had been screened.These results should be valuable for studying HCMV immunology mechanism, effect of viral genes on cells and new vaccines.