HBV核酸定量检测试剂盒的研发及T细胞免疫水平的研究
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摘要
乙型肝炎病毒（HBV）为一种流行十分广泛的部分双链DNA病毒，是导致急慢性肝炎和肝细胞癌变的致病因子，严重危害了人类公共卫生安全。目前，全世界有将近20亿人感染过HBV，其中慢性感染者约为3.5亿。中国HBV感染率为7.18%，大约有1亿人为HBV感染者。慢性HBV感染者并不表现有任何临床症状，但具有较强的传染性并有可能发展为肝硬化及肝癌等严重疾病。因此检测筛查HBV是否感染、感染后的治疗效果评价以及免疫水平监测是一项艰巨的工程。
目前以检测HBsAg为阳性作为HBV感染的标志。然而在慢性感染的病人中有一部分人群针对HBsAg产生的细胞免疫很弱，而且有的几乎检测不到抗原及相应的抗体。这部分HBsAg阴性的HBV感染者可以用核酸定量的检测方法检出，这就需要有可靠的HBV DNA检测技术。而在急性HBV感染的病人中几乎都能够针对HBsAg、HBcAg产生强烈的细胞免疫响应。在体外检测T细胞免疫响应需要具有抗原投递功能的载体将抗原递呈给特异性T细胞。
本研究中，为了开发高特异性、高灵敏性的HBV DNA荧光定量PCR检测试剂盒及研究HBV感染者机体T细胞免疫水平，我们设计了以下两部分试验。第一部分：针对HBV不同基因型A～H高度保守区设计4对引物(HBsF1/HBsR1、HBsF2/HBsR2、HBxF/HBxR、HBcF/HBcR)和相对应的4条Taqman探针（HBsP1、HBsP2、HBxP、HBcP）。通过用4对引物对HBV DNA扩增并与T载体连接后，构建了pMD18-T-HBs1、pMD18-T-HBs2、pMD18-T-HBx和pMD18-T-HBc4种重组质粒，并计算拷贝数后作为荧光定量PCR检测HBV DNA方法的工作标准品。经荧光定量PCR检测分析发现，pMD18-T-HBx和pMD18-T-HBc在1.0×103copies/mL～1.0×109copies/mL时，标准曲线的线性范围相关系数R2为0.99以上，扩增效率为95%～105%，较pMD18-T-HBs1、pMD18-T-HBs2的线性范围宽，扩增效率高。以pMD18-T-HBc为工作标准品对荧光定量PCR检测HBV DNA的方法学进行评价，批内精密度为0.12%～1.01%，批间精密度为1.99%～7.01%。该方法最低检出限为125copies/mL，比市售同类商品检出限低4～8倍。对HBV核酸国家标准品中阳性标准品、阴性标准品符合率均为100%，对国家线性标准品检测的线性相关系数R2为0.9992。对不同类型的血样检测发现，经EDTA抗凝的血浆检测值要高于血清检测值，肝素钠抗凝的血浆检测值效果最差。对用试剂盒和热裂解提取血浆HBV DNA检测发现，试剂盒提取病毒载量较低的样品时，效果要好于热裂解法。对102份HBsAg（+）血浆检测并与HBV商品化检测试剂盒比较分析，总符合率为94.1%，相关系数R2为0.951。
第二部分：将含有抗原投递系统LFn的核心抗原肽库重组质粒转化入表达菌株E.coli BL21后，经原核表达、IMAC亲和层析、QFF离子交换层析、超滤等方法进行纯化、去内毒素、浓缩并更换缓冲液处理。所得蛋白对HBV感染者全血体外刺激，并经ELISA检测IFN-γ含量。分析发现,对于经核心肽库抗原刺激的血浆中IFN-γ含量，病毒载量大于105copies/mL组明显高于病毒载量小于105copies/mL组。HBsAg（+）、HBeAg（+）、HBcAb（+）组明显高于HBsAg（+）、HBeAb（+）、HBcAb（+）组。经T检验分析，以上两组IFN-γ含量差异显著。
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.
目前以检测HBsAg为阳性作为HBV感染的标志。然而在慢性感染的病人中有一部分人群针对HBsAg产生的细胞免疫很弱，而且有的几乎检测不到抗原及相应的抗体。这部分HBsAg阴性的HBV感染者可以用核酸定量的检测方法检出，这就需要有可靠的HBV DNA检测技术。而在急性HBV感染的病人中几乎都能够针对HBsAg、HBcAg产生强烈的细胞免疫响应。在体外检测T细胞免疫响应需要具有抗原投递功能的载体将抗原递呈给特异性T细胞。
本研究中，为了开发高特异性、高灵敏性的HBV DNA荧光定量PCR检测试剂盒及研究HBV感染者机体T细胞免疫水平，我们设计了以下两部分试验。第一部分：针对HBV不同基因型A～H高度保守区设计4对引物(HBsF1/HBsR1、HBsF2/HBsR2、HBxF/HBxR、HBcF/HBcR)和相对应的4条Taqman探针（HBsP1、HBsP2、HBxP、HBcP）。通过用4对引物对HBV DNA扩增并与T载体连接后，构建了pMD18-T-HBs1、pMD18-T-HBs2、pMD18-T-HBx和pMD18-T-HBc4种重组质粒，并计算拷贝数后作为荧光定量PCR检测HBV DNA方法的工作标准品。经荧光定量PCR检测分析发现，pMD18-T-HBx和pMD18-T-HBc在1.0×103copies/mL～1.0×109copies/mL时，标准曲线的线性范围相关系数R2为0.99以上，扩增效率为95%～105%，较pMD18-T-HBs1、pMD18-T-HBs2的线性范围宽，扩增效率高。以pMD18-T-HBc为工作标准品对荧光定量PCR检测HBV DNA的方法学进行评价，批内精密度为0.12%～1.01%，批间精密度为1.99%～7.01%。该方法最低检出限为125copies/mL，比市售同类商品检出限低4～8倍。对HBV核酸国家标准品中阳性标准品、阴性标准品符合率均为100%，对国家线性标准品检测的线性相关系数R2为0.9992。对不同类型的血样检测发现，经EDTA抗凝的血浆检测值要高于血清检测值，肝素钠抗凝的血浆检测值效果最差。对用试剂盒和热裂解提取血浆HBV DNA检测发现，试剂盒提取病毒载量较低的样品时，效果要好于热裂解法。对102份HBsAg（+）血浆检测并与HBV商品化检测试剂盒比较分析，总符合率为94.1%，相关系数R2为0.951。
第二部分：将含有抗原投递系统LFn的核心抗原肽库重组质粒转化入表达菌株E.coli BL21后，经原核表达、IMAC亲和层析、QFF离子交换层析、超滤等方法进行纯化、去内毒素、浓缩并更换缓冲液处理。所得蛋白对HBV感染者全血体外刺激，并经ELISA检测IFN-γ含量。分析发现,对于经核心肽库抗原刺激的血浆中IFN-γ含量，病毒载量大于105copies/mL组明显高于病毒载量小于105copies/mL组。HBsAg（+）、HBeAg（+）、HBcAb（+）组明显高于HBsAg（+）、HBeAb（+）、HBcAb（+）组。经T检验分析，以上两组IFN-γ含量差异显著。
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.
引文
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