HBV与HCV融合基因免疫的实验研究
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摘要
在病毒性肝炎中，乙型病毒性肝炎和丙型病毒性肝炎对人类健康危害最
为严重，二者均可通过输血、伤口、及密切接触传播，引起急性和慢性肝炎。
我国每年死于肝病的患者达20万人，其中70％以上与乙型病毒性肝炎和丙型
病毒性肝炎相关。两者的发病有一定的相似性及重叠性，HBsAg（HBV表面抗
原）阳性的肝细胞癌患者HCV阳性率高达20％～30％，而且HCV与HBV可协同
作用而导致更为严重的肝损害，其预后比单纯的HBV或HCV感染更差，HBV
和HCV重叠感染引起的肝硬变发生肝细胞癌（HCC）机率（48％）比非这些
病毒引起肝硬变者发生HCC的机率（13％）要高得多。目前虽有乙型肝炎疫
苗使用，但还着存在免疫保护维持时间短及部分病人对疫苗免疫反应水平低
下等缺点。丙型肝炎病毒型别多，包膜糖蛋白高度变异，目前尚未研制出蛋
白疫苗。因此构建能同时预防HBV与HCV感染的新型疫苗，将有特别意义。
近年来发展起来的基因免疫为预防HBV和HCV感染带来了新的希望。基
因免疫又称核酸免疫，是指将含有目的基因编码序列的真核表达质粒，直接
导入宿主体内，以诱导宿主免疫系统对目的基因所表达的外源蛋白发生特异
性免疫应答，达到免疫的目的。与传统蛋白疫苗相比，基因免疫具有易制备、
操作简便、不需纯化抗原蛋白并能同时诱导体液免疫和细胞免疫应答等优
势。基因疫苗研究中发现，仅有病毒核心抗原的编码基因虽可产生抗体应答，
但一般不能产生具有中和作用的抗体应答，所以近年都采用核心抗原基因与
第口旱匡大学协士学位论文
一
iM抗原基因或与llBV表面抗原基因相触合，以讪同时获得抗HCV和HBV的体
液免疫和细胞兔疫。
HBV为嗜肝洲A病毒，其编码的包膜蛋白山 HBsAs、PreSIAs和 ＂res 耶s
组成，中和表位位于S区，是目前应用的乙肝疫苗的主要成份。PreslAg和
preSZAg分别含有不同于HBsAs抗原的B细胞表位和T细胞表位。目前对于
preSIAg和／或PreSZAe是否可改进现用疫苗的保护性尚无定论，然而有些研
究结果提示 preslAg和／或 preSZAg可提供促进产生抗 HBs的 Th细胞作用，
从而使遗传性的低反应或无反应机体产生抗体。HCV为单股正链RNA病毒，
其疫苗的研究主要集中在核心蛋白C区和包膜蛋白【区。在HCV基因组中，
C区是最为保守的区域，并且含有至少5个重要的T细胞和B细胞表位，该
区所表达的核心蛋白虽不能诱导中和抗体，却是机体特异性CTL的重要靶抗
原，具有极强的兔疫原性。因此包含C区的HCV核酸疫苗可能诱导高水平特
异性抗体应答及CTL活性，其中又以CTL活性在HCV核酸疫苗免疫应答作用
中最有意义。CTL在HCV的感染过程中可发挥其细胞毒作用并最终清除感染
细胞，这将有助于克服HCV的高变异性及机体对HCV蛋白质疫苗的无应答性。
有鉴于此，我们构建了HBV与HCV的融合基因疫苗，并对其诱导的体液
免疫与细胞免疫应答进行了研究，主要内容包括：
1．应用分子克隆技术，构建了同时或分别含HBV S… 6或
preSZ侣）基因与 HCV C区基因的真核表达质粒：CSpcDNA3．1、CSlpCDNA3．1、
CSZpcDNA3．1、CpcDNA3．l和SpcDNA3．1。
2．将真核表达质粒CSpcDNA3．l用电穿孔法转染与Balb／c ．J’鼠基因型
相同的 PS 15细胞。转染细胞经 G4筛选后，再经有限稀释法克隆筛选，获
得了能同时表达HBV表面抗原与HCV核心蛋白的稳定细胞系。
3．比较CSP。DNA3．1、CSIPCDNA3．l、CSZPCDNA3．1三种融合重组质粒的
免疫效果。结果发现，两次免疫后，全部小鼠均产生了抗HBS和抗HCV，且
·4·
第回罩巨大学协士学位论文
一
抗 HCV的产生水平高于抗HBs；CSlpcDNA3．l和 CSZpcDNA3．1翩免疫小鼠，
产生抗HBS的水平低于CSpCDNA3．l，说明pr妨基因对抗HBS的产生有负调
控作用；CSZpcDNA3．l和CSpcDNA3．l质粒免疫的小鼠，产生的抗HCV水平高
于CSlpcDNA3．l，说明presl基因对抗HCV的产生也存在着负调控作用。
4．研究了不同注射剂量，不同免疫次数、脂质体包裹、HBsAg和HCcAg
（HCV核心抗原）加强注射等条件下，对CSPCDNA3．1质粒免疫效果的影响。
结果发现，在一定范围内，DNA兔疫应答强度与剂量和接种次数呈相关性，
1mg和3次免疫分别为最佳剂量和次数：脂质体对抗册s和抗＊V的阳转
率无明显影响，但能提高应答强度；HBSAg和HCCAg蛋白抗原?
Hepatitis B and hepatitis C are major diseases in virus hepatitis. Both of
them can be transmitted by transfusion, wound and nearly touch. Hepatitis B and
hepatitis C include acute and chronic hepatitis. Over 200 thousand peoples cued
of liver diseases every year and 70 percent of them was correlative with hepa~tis
B and hepatitis C. Peoples can be infected by overlapped hepatitis B virus and
hepatitis C virus. Anti-HCV is 20 to 30 percent in hepatocellar carcinoma(HCC)
with HBsAg. In hepaocirrhosis, the rate of HCC with overlaping infection is
much higher than that of non-B and non-C. HBV and HCV can interact and result
in servious liver damage. The prognosis is worst than HBV or HCV infection.
Though HBV vaccine has been used over the world, the protetion period of HBV
infection is not enough long and the immune response is weak in some peoples.
No vaccine has been used until now because of the high gene mutant in HCV. So
it is important to contruct chimeric genetic vaccine of HBV and HCV.
Genetic immunization (nucleic acid immunization) has emerged recently and
has brought new hope for HBV and HCV infection. It is a promising novel
strategy that naked DNA encoding spcific antigens and the regulatory elements
are introduced directly into niamnialian tissue, and subsequently induce specific
humoral and cellular immune responses to the expressed antigen. It抯
significantly cheaper to manufacture than recombinant polypeptide. The
considerable costs involved in producing and purifying protein antigens would be
avoided. It is found that only including virus core protein can induce antibody
.6-
after immunization but has no neutralization . So recently chimric HI3V envelope
and HCV core gene contructs is generally used.
HBV belongs to DNA virus known as hepadna viridae. The envelope open
reading frame contains three in-phase translation start condons that divide the
gene into three coding regions known as preS I, preS2 and 5, in which the major
neutralizing antigen epitope locates. The commercially available hepatitis B
vaccines presently used have mainly consisted of the S protein. It has shown that
the B and T cell epitopes in preS 1 and preS2 are different from S antigen. It is not
sure whether the preS 1 and preS2 antigens can improve protection of present
vaccines. However, some researches suggest that they can enchance helper T cell
to produce antibody. HCV belongs to single positive-strand RNA virus. Vaccine
reearches has concentrated on HCV eore(C) protein and envelope(E)
glycoproteins. The core gene shows greater sequence conservation among HCV
strains and the core protein includes at least 5 epitopes of B and T cell. The
immunogenicity of core protein is the strongest among the structural proteins and
it is the most important target antigen for Cytotoxic T lyphocyte (CTL). So, the
genetic immunization including C gene offers the potential advantage of inducing
cellar immune responses. Morever, these cellar immune responses may also have
the benefit of eradicating persistent viral infection from the host and help to
overcome the low response to HCV protein vaccine because of the high mutant.
On the basis of all these study, we contructed the chimeric genetic vaccine of
HBV and HCV and investigated the humoral and cellular immune responses after
genetic immunization.
The stuidies consisted of:
1. Five recombinant eukaryotic expression plasmids were construc
为严重，二者均可通过输血、伤口、及密切接触传播，引起急性和慢性肝炎。
我国每年死于肝病的患者达20万人，其中70％以上与乙型病毒性肝炎和丙型
病毒性肝炎相关。两者的发病有一定的相似性及重叠性，HBsAg（HBV表面抗
原）阳性的肝细胞癌患者HCV阳性率高达20％～30％，而且HCV与HBV可协同
作用而导致更为严重的肝损害，其预后比单纯的HBV或HCV感染更差，HBV
和HCV重叠感染引起的肝硬变发生肝细胞癌（HCC）机率（48％）比非这些
病毒引起肝硬变者发生HCC的机率（13％）要高得多。目前虽有乙型肝炎疫
苗使用，但还着存在免疫保护维持时间短及部分病人对疫苗免疫反应水平低
下等缺点。丙型肝炎病毒型别多，包膜糖蛋白高度变异，目前尚未研制出蛋
白疫苗。因此构建能同时预防HBV与HCV感染的新型疫苗，将有特别意义。
近年来发展起来的基因免疫为预防HBV和HCV感染带来了新的希望。基
因免疫又称核酸免疫，是指将含有目的基因编码序列的真核表达质粒，直接
导入宿主体内，以诱导宿主免疫系统对目的基因所表达的外源蛋白发生特异
性免疫应答，达到免疫的目的。与传统蛋白疫苗相比，基因免疫具有易制备、
操作简便、不需纯化抗原蛋白并能同时诱导体液免疫和细胞免疫应答等优
势。基因疫苗研究中发现，仅有病毒核心抗原的编码基因虽可产生抗体应答，
但一般不能产生具有中和作用的抗体应答，所以近年都采用核心抗原基因与
第口旱匡大学协士学位论文
一
iM抗原基因或与llBV表面抗原基因相触合，以讪同时获得抗HCV和HBV的体
液免疫和细胞兔疫。
HBV为嗜肝洲A病毒，其编码的包膜蛋白山 HBsAs、PreSIAs和 ＂res 耶s
组成，中和表位位于S区，是目前应用的乙肝疫苗的主要成份。PreslAg和
preSZAg分别含有不同于HBsAs抗原的B细胞表位和T细胞表位。目前对于
preSIAg和／或PreSZAe是否可改进现用疫苗的保护性尚无定论，然而有些研
究结果提示 preslAg和／或 preSZAg可提供促进产生抗 HBs的 Th细胞作用，
从而使遗传性的低反应或无反应机体产生抗体。HCV为单股正链RNA病毒，
其疫苗的研究主要集中在核心蛋白C区和包膜蛋白【区。在HCV基因组中，
C区是最为保守的区域，并且含有至少5个重要的T细胞和B细胞表位，该
区所表达的核心蛋白虽不能诱导中和抗体，却是机体特异性CTL的重要靶抗
原，具有极强的兔疫原性。因此包含C区的HCV核酸疫苗可能诱导高水平特
异性抗体应答及CTL活性，其中又以CTL活性在HCV核酸疫苗免疫应答作用
中最有意义。CTL在HCV的感染过程中可发挥其细胞毒作用并最终清除感染
细胞，这将有助于克服HCV的高变异性及机体对HCV蛋白质疫苗的无应答性。
有鉴于此，我们构建了HBV与HCV的融合基因疫苗，并对其诱导的体液
免疫与细胞免疫应答进行了研究，主要内容包括：
1．应用分子克隆技术，构建了同时或分别含HBV S… 6或
preSZ侣）基因与 HCV C区基因的真核表达质粒：CSpcDNA3．1、CSlpCDNA3．1、
CSZpcDNA3．1、CpcDNA3．l和SpcDNA3．1。
2．将真核表达质粒CSpcDNA3．l用电穿孔法转染与Balb／c ．J’鼠基因型
相同的 PS 15细胞。转染细胞经 G4筛选后，再经有限稀释法克隆筛选，获
得了能同时表达HBV表面抗原与HCV核心蛋白的稳定细胞系。
3．比较CSP。DNA3．1、CSIPCDNA3．l、CSZPCDNA3．1三种融合重组质粒的
免疫效果。结果发现，两次免疫后，全部小鼠均产生了抗HBS和抗HCV，且
·4·
第回罩巨大学协士学位论文
一
抗 HCV的产生水平高于抗HBs；CSlpcDNA3．l和 CSZpcDNA3．1翩免疫小鼠，
产生抗HBS的水平低于CSpCDNA3．l，说明pr妨基因对抗HBS的产生有负调
控作用；CSZpcDNA3．l和CSpcDNA3．l质粒免疫的小鼠，产生的抗HCV水平高
于CSlpcDNA3．l，说明presl基因对抗HCV的产生也存在着负调控作用。
4．研究了不同注射剂量，不同免疫次数、脂质体包裹、HBsAg和HCcAg
（HCV核心抗原）加强注射等条件下，对CSPCDNA3．1质粒免疫效果的影响。
结果发现，在一定范围内，DNA兔疫应答强度与剂量和接种次数呈相关性，
1mg和3次免疫分别为最佳剂量和次数：脂质体对抗册s和抗＊V的阳转
率无明显影响，但能提高应答强度；HBSAg和HCCAg蛋白抗原?
Hepatitis B and hepatitis C are major diseases in virus hepatitis. Both of
them can be transmitted by transfusion, wound and nearly touch. Hepatitis B and
hepatitis C include acute and chronic hepatitis. Over 200 thousand peoples cued
of liver diseases every year and 70 percent of them was correlative with hepa~tis
B and hepatitis C. Peoples can be infected by overlapped hepatitis B virus and
hepatitis C virus. Anti-HCV is 20 to 30 percent in hepatocellar carcinoma(HCC)
with HBsAg. In hepaocirrhosis, the rate of HCC with overlaping infection is
much higher than that of non-B and non-C. HBV and HCV can interact and result
in servious liver damage. The prognosis is worst than HBV or HCV infection.
Though HBV vaccine has been used over the world, the protetion period of HBV
infection is not enough long and the immune response is weak in some peoples.
No vaccine has been used until now because of the high gene mutant in HCV. So
it is important to contruct chimeric genetic vaccine of HBV and HCV.
Genetic immunization (nucleic acid immunization) has emerged recently and
has brought new hope for HBV and HCV infection. It is a promising novel
strategy that naked DNA encoding spcific antigens and the regulatory elements
are introduced directly into niamnialian tissue, and subsequently induce specific
humoral and cellular immune responses to the expressed antigen. It抯
significantly cheaper to manufacture than recombinant polypeptide. The
considerable costs involved in producing and purifying protein antigens would be
avoided. It is found that only including virus core protein can induce antibody
.6-
after immunization but has no neutralization . So recently chimric HI3V envelope
and HCV core gene contructs is generally used.
HBV belongs to DNA virus known as hepadna viridae. The envelope open
reading frame contains three in-phase translation start condons that divide the
gene into three coding regions known as preS I, preS2 and 5, in which the major
neutralizing antigen epitope locates. The commercially available hepatitis B
vaccines presently used have mainly consisted of the S protein. It has shown that
the B and T cell epitopes in preS 1 and preS2 are different from S antigen. It is not
sure whether the preS 1 and preS2 antigens can improve protection of present
vaccines. However, some researches suggest that they can enchance helper T cell
to produce antibody. HCV belongs to single positive-strand RNA virus. Vaccine
reearches has concentrated on HCV eore(C) protein and envelope(E)
glycoproteins. The core gene shows greater sequence conservation among HCV
strains and the core protein includes at least 5 epitopes of B and T cell. The
immunogenicity of core protein is the strongest among the structural proteins and
it is the most important target antigen for Cytotoxic T lyphocyte (CTL). So, the
genetic immunization including C gene offers the potential advantage of inducing
cellar immune responses. Morever, these cellar immune responses may also have
the benefit of eradicating persistent viral infection from the host and help to
overcome the low response to HCV protein vaccine because of the high mutant.
On the basis of all these study, we contructed the chimeric genetic vaccine of
HBV and HCV and investigated the humoral and cellular immune responses after
genetic immunization.
The stuidies consisted of:
1. Five recombinant eukaryotic expression plasmids were construc
引文
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