| AIM: 11EV is the cause of hepatitis B, its genome shows higher nucleotide sequence variability. Lots of HBsAg carriers bave various HBV strains(heterogeneity). Different strains have distinct replication level, immunity and ability of transmission, which have different effects on the developments and outcomes of the disease. In order to explore the variability of 11EV genome in HBsAg positive pregnant women and the relationship between the variability and intrauterine transmission, we established a method to amplify the full-length HBV genome. By this method, the whole genome of 11EV strains from sera samples of patients was obtained. In addition, we sequenced and analyzed the preS/S gene in HBsAg positive pregnant women and their new-borns. METHODS: ?HBV DNA positive sera of 10 patients at Xijing hospital were collected from Jan 桱une, 2000. HBV DNA was extracted from the sera. Then we set up a full-length PCR and the condition of the PCR were optimized. ?Purified PCR products, cloned it to the vector, got the full-length HEV genomes, identified them using PCR, restriction endonuclease, sequencing at the same time. Fifty HBsAg positive pregnant women and their newborns were selected as subjects between 1997?998 in Taiyuan infectious hospital and blood specimens from those mothers and infants at birth within 24 hours by femoral puncture were collected. Used the new method to amplify the full-length HBV genome of 8 pregnant women successfully and the whole HBV clones were obtained, then amplified preS/S gene and analyzed the sequence by MEGL17NE and VECTER NTI software. ?Amplified preS/S gene of 2 newborns, compared relevant sequences with their mothers, other HBsAg positive pregnant women and other HBV infected patients. Results: (D Establishment of 11EV full-length PCR method: By optimized the condition of PCR, the best PCR systems as follows: l0xBuffer 4.5 jil, 25mM MgC12 3pi, 72mM dNTP 1j.d, each primer: Pi. P2 3OpM, template 5p.l, WO 45j.tl, 3 drops oil. The reactions w. nut as a ot start?PCR, A 451.L1 reaction premix was heated to 850, and 5j.il enzyme mix (Taq/Pwo DNA polymerase mix 2.6u, lOxBuffer 0.5j.d, H20 )was then added, the best PCR cycle: 940 45sec, 600 1mm, 720 4mm with an increment of 5 s/cycle for elongation, after 40 cycles,72 0 1 0mm. By new method, 3 out of 10 samples appeared the amplified 3.2kb fragments,all 3 sera samples were HBsAg,HBeAg and anti-HBc positive and all plasmid appeared expected PCR products.Tbis method was steady, clear and trusty. Recombinant HBV plasmid was identified. ?We used the full-length PCR having amplified I{BV genome of 8 HBsAg positive pregnant women successfully. Four women抯 neonates infected with HEY, and the other's neonates didn't infect with. We selected 5 whole 11EV clones form each sample and analyzed their preS/S gene. All nucleotide sequences contribute to HBV C genome type. Average base substitution rate of 20 clones was 3.50%, lack rate was 0.70%, insert rate was 1.21% in 4 HBsAg positive pregnant women whose neonates infected 11EV. Compared with 4 HBsAg positive pregnant women, whose neonates didn't infect with HBV, the average base substitution rate, lack rate and insert rate of 20 clones were2.27%, 0.20/s and 0.26% respectively. The most popular mutation type was substitution. There is a statistical difference between the two groups(y=11 and P<0.00 1). The mutation of preS/S gene also showed higher variability o... |
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