| Hepatitis B virus (HBV) is a double-stranded DNAvirus which is spread all over theworld. It can lead to the acute/chronic hepatitis and hepatic carcinoma, and seriouslyendangering the public health security. Currently, nearly20million people in worldwideinfected with HBV, and about350million people were chronic carriers. HBV infectionrate was7.18%in China. Most Chronic HBV carriers show no clinical symptoms, butthey have a strong infectious and may developing into cirrhosis and hepatic carcinoma.Therefore, there is a difficult task for detection of HBV infection screening, evaluation oftreatment after infection and the immune level monitoring.Currently, the sign of HBV infection is test positive for HBsAg. However, someonewho is chronic carriers is weak against HBsAg by cellular immunity and could not bedetected HBV antigens and antibodies. These HBV carriers who are the HBsAg-negativecan be detected using real time PCR. Almost all HBV acute patient can generate strongimmune response for HBsAg and HBcAg. Detection T cell immune function in vitroneeds to antigen delivery carrier which can presenting specific antigen to T cells.In this study, in order to develop a HBV DNA quantitative PCR detection kit forhigh specificity and high sensitivity, and research T cell immunity of HBV carriers, wedesigned the following two-part test. Part I: designed four pairs of primers(HBsF1/HBsR1, HBsF2/HBsR2, HBxF/HBxR, HBcF/HBcR) in highly conservedarray of HBV genotypes A~H, and the corresponding four Taqman probe (HBsP1,HBsP2, HBxP, HBcP). Amplified HBV DNAusing four pairs of primers, and connectedwith the T vector to constructed four kinds of recombinant plasmid (pMD18-T-HBs1,pMD18-T-HBs2, pMD18-T-HBx and pMD18-T-HBc). The copy number of recombinantplasmid was calculated, and detected recombinant plasmid using the fluorescencequantitative PCR. The concentration of Recombinant plasmid pMD18-T-HBx andpMD18-T-HBc is1.0×103copies/mL to1.0×109copies/mL, the correlation coefficientof the standard curve linear range is0.99, amplification efficiency range was95%to105%. Compared with pMD18-T-HBs1, pMD18-T-HBs2, pMD18-T-HBx andpMD18-T-HBc have a wider linear range, and better amplification efficiency. As thework standard of real time PCR, intra-precision of pMD18-T-HBc is about0.12%to1.01%, inter-precision of pMD18-T-HBc is about1.99%to7.01%. The detection limit ofreal time PCR was125copies/mL. compare with other commercial products, thedetection limit of real time PCR is lower4to8times. Detected National Standard forHBV DNA, the coincidence rate of positive standard and negative standard was100%,correlation coefficient of the national linear standard and work standard is0.9992.Detected serum and plasma which is anticoagulant by EDTA and heparin, the viral loadof EDTA-plasma is higher than serum levels, heparin plasma is the worst. Detected HBVDNA in plasma which extract by Heat-lyses and virus DNA extraction kit, the viral loadof samples extract by kit is higher, the effect is better than the heat-lyses. Detection102 HBsAg (+) plasma and comparative analysis with commercial kit, the total coincidencerate was94.1%, correlation coefficient was0.951.Part II: recombinant plasmids of the core peptide library which contain LFn, aantigen delivery system, were transformed into E.coli BL21，The fusion proteins wereexpressed, and purification by IMAC affinity chromatography, wipe off endotoxin byQFF ion exchange chromatography, concentrate and exchange buffer by ultrafiltrationcolumn. Using HBcAg fusion proteins stimulate whole blood in vitro, and detected IFN-γby ELISA. The results showed that IFN-γ levels in plasma stimulating by core peptidesfusion proteins, the group of viral load greater than105copies/mL were significantlyhigher than the group of viral load less than105copies/mL group. The group of HBsAg(+), HBeAg (+), HBcAb (+) was significantly higher than the group of HBsAg (+),HBeAb (+), HBcAb (+). And these two groups of IFN-γ levels analysis by T test analysiswere significantly different. |
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