摘要
目 的
HBV具备改造作为肝靶向性基因治疗载体的基本条件：天然嗜肝特异性；能够在肝细胞内持续复制、反复感染，病毒本身对细胞无明显细胞毒性；能够携带外源基因并被包装成病毒颗粒，介导目的基因转移和表达。但因基因结构复杂，目前改造的HBV载体系统均存在载容量小、复制包装效率低及安全性差等缺点。基于此，本课题借鉴体内天然HBV变异株的生物学特性，优化HBV载体改造策略，探索性构建C基因截短型HBV载体，并对其表达、复制、包装等分子生物学特性进行探讨。
方 法
1. 用外源GFP报告基因取代野生HBV质粒pHBV3091 S基因编码区，同时保留S基因前9个碱基与GFP融合，构建携带外源报告基因的HBV载体pHBV-S-GFP。应用脂质体单独转染该HBV重组体，以GFP表达阳性质粒pCMV-GFP作对照，荧光显微镜下观察GFP在肝细胞中表达。重组体pHBV-S-GFP与缺失包装信号ε的辅助质粒pHBV3142共转染HepG2细胞，提取细胞核中病毒DNA，根据HBV 基因组复制特点设计包含GFP及HBV DNA序列在内的特异性引物，半巢式PCR检测HBV重组体复制中间体ccc DNA的生成；提取胞外上清液中病毒颗粒DNA，对同一样本依次PCR检测GFP基因及HBV DNA，验证分泌到上清液中携带外源基因的“治疗性”子代病毒颗粒生成；Southern blot检测重组HBV载体的包装效率，并与野生HBV质粒对照组进行比较。
以野生型HBV质粒pHBV3091为骨架，通过基因定点突变技术构建C基因截短型HBV突变体：pHBV-CΔ188（缺失220-407nt）和pHBV-CΔ141（缺失267-407nt）。应用脂质体单独转染或与缺失包装信号ε的辅助质粒pHBV3142共转染HepG2细胞，提取细胞浆及细胞培养上清液中病毒颗粒DNA进行Southern blot，检测C基因截短型HBV突变体的复制包装能力；提取细胞核中病毒DNA，根据HBV 基因组复制特点设计特异性引物，PCR检测C基因截短型HBV突变体复制中间体ccc DNA的生成；通过实时荧光定量PCR对胞外上清液中病毒颗粒进行定量分析，比较C基因截短型HBV突变体与野生型HBV的包装效率。以野生HBV质粒pHBV3091
2. 作对照，单独转染C基因截短型HBV突变体质粒，Trizol法提取转染细胞总RNA，荧光定量RT-PCR定量分析C基因截短对HBV RNA转录水平的影响；提取细胞浆中病毒颗粒，Western blot检测S蛋白的表达，ELISA方法定量分析C基因截短对S蛋白表达的影响。
3. 用外源GFP报告基因取代HBV S基因编码区（同方法1），构建C基因截短型（同方法2）HBV载体：pHBVΔC188-S-GFP与pHBVΔC141-S-GFP。应用脂质体分别转染HepG2细胞，以C基因非截短型HBV载体pHBV-S-GFP质粒作阳性对照，荧光显微镜下观察细胞中GFP表达。与缺失包装信号ε的辅助质粒pHBV3142共转染HepG2细胞，提取细胞核中病毒DNA，根据HBV 基因组复制特点设计同时包含GFP基因及HBV DNA序列在内的特异性引物，半巢式PCR检测该类HBV重组体复制中间体ccc DNA的生成；提取细胞浆中病毒颗粒进行病毒包装实验，验证C基因截短型HBV载体的包装能力：非变性 Western blot检测病毒颗粒的C蛋白， Southern blot检测C蛋白包裹的DNA；提取胞浆中病毒颗粒DNA，常规Southern blot后对杂交信号进行扫描，定量分析C基因截短型HBV载体的包装效率；提取上清液中病毒DNA，设计同时包含GFP基因片段及HBV DNA序列在内的特异性引物，PCR验证携带外源基因的“治疗性”子代病毒颗粒分泌到胞外上清液中。
结 果
1. 外源GFP报告基因取代HBV S基因读码框破坏了P基因结构的完整性，因此重组体pHBV-S-GFP自身复制能力丧失，为复制缺损型载体。单独转染HepG2细胞后，可在蓝光（488nm）激发下发出绿色荧光，但荧光强度相对弱于阳性对照。与缺失包装信号ε的辅助质粒pHBV3142共转染肝细胞，可在细胞核提取物中检测到含有GFP基因的HBV复制中间体ccc DNA生成；常规PCR检测发现，携带GFP基因的子代病毒颗粒能够分泌到胞外上清液中；进一步Southern blot证实，pHBV-S-GFP/ pHBV3142共转染后病毒包装效率明显低于野生HBV质粒pHBV3091。
HBV突变体pHBV-CΔ141 与pHBV-CΔ188的C基因截短变异均发生在P基因读码框5’近端附近，但P基因结构没有受到影响。其中前者为模拟体内天然变异株的基因结构构建，后者则在前者基础上进一步将C基因截短延长到Δ188nt。两者分别单独转染HepG2细胞，提取胞浆及上清液中病毒DNA进行Southern blot，杂交结果均阴性；后分别与缺失包装信号ε的辅助质粒pHBV3142共转染肝细胞，提取胞浆及上清液中病毒DNA
2. 进行Southern blot，两者杂交结果均阳性，可见到HBV DNA各种复制中间体杂交信号：如RC DNA、DS DNA、SS DNA等；通过特异性PCR可在细胞核内检测到HBV变异体ccc DNA的生成；与pHBV3091/pHBV3142转染组相比，C基因截短型HBV突变体的Southern blot杂交信号显著增强；进一步对培养上清液中子代病毒颗粒DNA进行定量：pHBV-CΔ188 / pHBV3142组为2.2×109copies/ml、pHBV-CΔ141 / pHBV3142组为1.5×108copies/ml、pHBV3091 / pHBV3142为4.8×107 copies/ml，提示C基因截短型HBV突变体的包装效率显著提高，其中C基因截短188nt后效果更为明显；荧光定量RT-PCR显示，C基因截短对HBV RNA的转录水平没有影响?
Objectives
Hepatitis B virus (HBV) is naturally provided with the prerequisite for being transformed as the liver-targeting gene therapeutic vector because of its hepatotropism specificity. It can specifically infect the liver and continuously replicate in the hepatocytes without obvious cytotoxicity by itself. Moreover, it has also the capability of carrying, transferring and expressing the gene of interest, which is ultimately encapsidated into the viral particles. However, it is a pity that there are many shortcomings in the present developed HBV-based vectors such as the poor capability of carrying long foreign gene, the low efficiency of replication and encapsidation, the potential risk of immunogenicity etc, which all result from the complex genomic structure of HBV. In view of the biological features of the naturally occurring defective HBV mutants obtained from the hepatitis patients, the C gene-truncated HBV vectors were experimentally constructed in this study as well as investigated for their molecular biological features including the expression, replication and encapsidation etc. in order to seek for the ideal strategy for developing the HBV-based vector.
Methods
The recombinant HBV vector of pHBV-S-GFP carrying the gene of interest was constructed in which a fragment encompassing the S gene was replaced by a DNA fragment encoding GFP fused to the first three amino acids of S. After the recombinant plasmid of pHBV-S-GFP was solely transfected to HepG2 by using the liposome method, the expression of GFP was observed with the fluorescence microscope. At the same time the plasmid of pCMV-GFP was selected as the positive control in parallel, which could effectively express the green fluorescence protein in the liver cells. After the plasmid of pHBV-S-GFP was co-transfected with the helper construct of pHBV3142 devoid of the encapsidation signal ε, the viral DNA were
1. extracted from the transfected HepG2 nucleolus about 48 hours later. The rHBV cccDNA was testified by using Semi-Nested-PCR with the specific primers designed to encompass both GFP and HBV DNA sequence in terms of the characteristic of HBV replication. The extract from culture medium was detected by PCR for both GFP and HBV DNA in order to testify the possibility that the "therapeutic" rHBV progeny particles carrying the GFP gene could be encapsidated to secrete into the supernatant. The encapsidation efficiency of the rHBV vector was evaluated by southern blot assay in which the wild plasmid pHBV3091 was used as control.
2. The C-truncated HBV mutants, pHBV-C△188( 220-407nt) and pHBV-C△141(267-407nt), were constructed by the molecular cloning and PCR-based deletion from the wild HBV plasmid of pHBV3091.The DNA of virus mutants were extracted from the cytoplasm and supernatant after the mutants plasmids were transfected solely or co-transfected with the helper construct of pHBV3142 devoid of the encapsidation signal ε.The replicating and encapsidating capabilities of both C-truncated HBV mutants were investigated by southern blot assay. These replication intermediate ccc DNA of both mutants extracted from the nucleolus of transfected HepG2 were testified by using Semi-Nested-PCR with the specific primers which were designed in terms of the characteristic of HBV replication. These mutants DNA extracted from the extracelluar supernatant were analyzed by using the Real-time fluorescence quantitative PCR in order to compare the encapsidation efficiency between the C-truncated HBV mutants and the wild HBV. The mutants RNA were extracted from the transfected HepG2 cells to evaluate quantitatively the effect on HBV RNA transcription in both C-truncated HBV mutants by using the Real-time fluorescence quantitative RT-PCR. The S protein expression was detected from the cytoplasmic lysates by western blot assay and further analyzed quantitatively by ELISA to determine the effect of the truncated C gene on S protein expression.
The C-truncated rHBV vectors carrying the reporter gene of GFP were constructed as pHBV-C△188-S-GFP and
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