单纯疱疹病毒Ⅱ型gD糖蛋白加趋化因子MIP-1α核酸疫苗的构建及初步免疫观察
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摘要
单纯疱疹病毒Ⅱ型(HSV-Ⅱ)主要引起生殖器疱疹。近年来，生殖器疱疹的发病率在迅速上升，危害性亦日益显得突出，但目前尚无特效药控制其发生和复发，因此研制疱疹病毒疫苗已成为预防生殖器疱疹感染的关键。在现已研制的疫苗中，DNA疫苗尽管诱导的免疫应答效力较低，可因其具有制备简单、安全、诱导的免疫应答广泛长久，并且可以共表达免疫调节因子，根据需要诱导产生最适免疫应答等特性，受到人们的青睐。近几年来，国外学者们一直在探索应用分子佐剂来加强DNA疫苗的免疫效果，并取得了一定的成效。为构建有效预防HSV-Ⅱ的DNA疫苗，探讨分子佐剂对DNA疫苗的作用机制，本研究完成了以下工作：
1．HSV-Ⅱ gD DNA疫苗的构建：根据GenBank中HSV-Ⅱ HG52株gD基因序列设计了一对引物，采用PCR方法扩增出HSV-Ⅱ Sav株gD全部编码区的基因片段，经中介pUCm-T载体，将其插入到真核表达质粒pcDNA3中相应的位点，并转化E．coli DH5α，构建出HSV-Ⅱ gD DNA疫苗。通过PCR、酶切和测序鉴定，证明gD基因片段在pcDNA3中插入正确，并将构建出的重组质粒命名为Pg。经原位ELISA法证实，Pg在COS-7细胞中瞬时表达的蛋白能够与gD单克隆抗体结合，这表明所构建的Pg DNA疫苗能够在哺乳动物细胞中表达。
2．小鼠趋化因子MIP-1α重组真核表达质粒的构建：首先用细菌脂多糖
天津医科大学博士研究生学位论文
(LPS)刺激RAW264.7细胞，提取总RNA，然后应用RT一PCR方法，扩增出MIP一1
。基因的全部编码序列。经中介pucm一T载体和Hindln、Xbal双酶切处理后，
将其插入到用相同酶切的真核表达质粒peDNA3中，并转化E.coh DHS“。通
过PCR、酶切和测序鉴定，证明MIP一la基因片段在PcDNA3中插入正确，
并将构建出的重组质粒命名为Pm。经RT一PCR和Boyden趋化小室法鉴定，证
实了所构建的Pm在哺乳动物细胞中能够表达具有生物学活性的MIP一1。。
3.pg+pm DNA疫苗对小鼠的初步免疫观察:每次用200林g/200林1 peDNA3、
Pg、Pg十Pm和等体积生理盐水(NS)肌肉注射免疫BALB/C小鼠，连续免疫3
次，末次免疫后一个月用IOOLDS。病毒量进行阴道内攻击。结果表明Pg+Pm疫
苗组的存活率(86.7%)明显高于Pg疫苗组的(53.3%);通过阴道组织病理切
片和半定量积分分析，证明了Pg+Pm疫苗比Pg疫苗明显降低了小鼠的发病率。
为探讨Pg+Pm DNA疫苗诱导的免疫应答，于末次免疫后第二周及第四周
收集小鼠血清和脾细胞，采用微量细胞中和试验和EL工SA法检测血清特异性抗
体，采用淋巴细胞转化试验检测脾T细胞增殖反应程度。结果显示Pg+Pm DNA
疫苗没有比Pg DNA疫苗诱导出更高的特异性抗体，但脾T细胞增值反应程度
明显高于Pg疫苗组的，表明Pg+Pm DNA疫苗诱导了更高的细胞免疫应答。
综上所述，本研究以HSv一H gD和M护一1。成功构建了预防HSv一H感染
的DNA疫苗。动物初步免疫发现，gD加MIP一1。的DNA疫苗免疫效应优于单
独的gD DNA疫苗，可以显著地提高小鼠的保护率和降低发病率。提示以
MIP一1。作为分子佐剂的HSV一H DNA疫苗具有潜在的应用价值。
Herpes simplex virus type II is the principal cause of genital herpes. In recent years, morbidity of genital herpes has been rising heavely, and its impairment becomes severe increasingly. But now there are no workable medicine to control its infection and recurrence. It is crucial to develop vaccine to prevent genital herpes. Although immune responses induced by DNA vaccine are lower, DNA vaccine is the most favorable among the developed vaccines, for it is safe and simple to make. Furthermore, DNA vaccine can effectively stimulate cell-mediated and humoral immune responses, and can coexpress with cytokines to induce the most appropriate immune responses on the basis of need. Recently, foreign scholars have been working for enhancing immune responses of DNA vaccine by using molecular adjuvant, and had got some achievement. In order to construct effective DNA vaccine to prevent HSV-II infection and explore modulation mechanisms of immunity of molecular adjuvant, we have made the following works in this study
: 1. Construction of DNA Vaccine of Herpes Simplex Virus Type II gD:
According to HSV-II strain HG52 gD gene sequence in GenBank, a pair of primers were designed and the whole encoding sequence of HSV-II strain Sav gD
was amplified by PCR. The PCR product was inserted into pUCm-T vector. Subsequently it was digested by restriction endonucleases HindIII and XbaI, and inserted orientally into mammlian vector pcDNA3 at corresponding sites. Thus the recombinant was transformed into E.coli DH5 a . Identified by PCR, restriction enzymes analysis and sequencing, recombinant eukaryotic expression plasmid was named Pg. When Pg was transiently transfected into COS-7 cell, specific recombinant protein gD was demonstrated by in situ ELISA with specific gD McAb. It indicated that Pg was able to express in mammalian cells.
2. Construction of recombinant eukaryotic expression plasmid of murine chemokine MIP-1α
The total RNA was extracted from LPS-stimulated RAW264.7 cell before the whole encoding sequence of MIP-1 a was amplified by RT-PCR. The RT-PCR product was inserted into pUCm-T vector. After RT-PCR product was digested by restriction endonucleases Hindlll and Xbal in pUCm-T vector, it was inserted orientally into mammlian vector pcDNA3 at corresponding sites. Then the recombinant was transformed into E.coli DH5 a . Identified by PCR, restriction enzymes analysis and sequencing , recombinant eukaryotic expression plasmid was named Pm. Specific recombinant protein rMIP-1α transiently expressed in COS-7 cell was demonstrated by RT-PCR and boyden chemotaxis chamber. It suggested that Pm be able to express in mammalian cells.
3. Assessment for protective effects of Pg +Pm DNA vaccine on mice
Mice were injected in the quadriceps with 200ug Pg, Pg+Pm in a final volume of 200ul of NS separately. The control mice were immunized with pcDNA3 and NS, respectively. Immunization was made for three times. One month after the last immunization, mice were challenged intravaginally with 100LD50 of HSV-II strain
Sav. Mice were then examined daily to evaluate pathological conditions and survival rates within 14 days. The results showed that Pg+Pm DNA vaccine improved, the survival rate of mice and reduced both the number of mice with lesions and severity of herpetic lesions significantly. 4. Evaluation of the immune responses induced by Pg +Pm DNA vaccine:
To detect the specific antibody titers by ELISA and microneutralization test, we collected serum at the second week and the fouth week after the last immunization. Lymphocyte transformation test (LTT) was made using MTT in the first month after the last immunization. Pg+Pm DNA vaccine resulted in a moderate but not significant enhancement of gD-specific IgG levels and induced significant enhancement of Th-cell proliferation responses. These results illustrated that Pg+Pm DNA vaccine induced higher antigen-specific cell-mediated immune responses than Pg DNA vaccine alone.
In sum, we have successfully constructed the DNA vaccine of HSV-II strain Sav gD with MIP-1α
1．HSV-Ⅱ gD DNA疫苗的构建：根据GenBank中HSV-Ⅱ HG52株gD基因序列设计了一对引物，采用PCR方法扩增出HSV-Ⅱ Sav株gD全部编码区的基因片段，经中介pUCm-T载体，将其插入到真核表达质粒pcDNA3中相应的位点，并转化E．coli DH5α，构建出HSV-Ⅱ gD DNA疫苗。通过PCR、酶切和测序鉴定，证明gD基因片段在pcDNA3中插入正确，并将构建出的重组质粒命名为Pg。经原位ELISA法证实，Pg在COS-7细胞中瞬时表达的蛋白能够与gD单克隆抗体结合，这表明所构建的Pg DNA疫苗能够在哺乳动物细胞中表达。
2．小鼠趋化因子MIP-1α重组真核表达质粒的构建：首先用细菌脂多糖
天津医科大学博士研究生学位论文
(LPS)刺激RAW264.7细胞，提取总RNA，然后应用RT一PCR方法，扩增出MIP一1
。基因的全部编码序列。经中介pucm一T载体和Hindln、Xbal双酶切处理后，
将其插入到用相同酶切的真核表达质粒peDNA3中，并转化E.coh DHS“。通
过PCR、酶切和测序鉴定，证明MIP一la基因片段在PcDNA3中插入正确，
并将构建出的重组质粒命名为Pm。经RT一PCR和Boyden趋化小室法鉴定，证
实了所构建的Pm在哺乳动物细胞中能够表达具有生物学活性的MIP一1。。
3.pg+pm DNA疫苗对小鼠的初步免疫观察:每次用200林g/200林1 peDNA3、
Pg、Pg十Pm和等体积生理盐水(NS)肌肉注射免疫BALB/C小鼠，连续免疫3
次，末次免疫后一个月用IOOLDS。病毒量进行阴道内攻击。结果表明Pg+Pm疫
苗组的存活率(86.7%)明显高于Pg疫苗组的(53.3%);通过阴道组织病理切
片和半定量积分分析，证明了Pg+Pm疫苗比Pg疫苗明显降低了小鼠的发病率。
为探讨Pg+Pm DNA疫苗诱导的免疫应答，于末次免疫后第二周及第四周
收集小鼠血清和脾细胞，采用微量细胞中和试验和EL工SA法检测血清特异性抗
体，采用淋巴细胞转化试验检测脾T细胞增殖反应程度。结果显示Pg+Pm DNA
疫苗没有比Pg DNA疫苗诱导出更高的特异性抗体，但脾T细胞增值反应程度
明显高于Pg疫苗组的，表明Pg+Pm DNA疫苗诱导了更高的细胞免疫应答。
综上所述，本研究以HSv一H gD和M护一1。成功构建了预防HSv一H感染
的DNA疫苗。动物初步免疫发现，gD加MIP一1。的DNA疫苗免疫效应优于单
独的gD DNA疫苗，可以显著地提高小鼠的保护率和降低发病率。提示以
MIP一1。作为分子佐剂的HSV一H DNA疫苗具有潜在的应用价值。
Herpes simplex virus type II is the principal cause of genital herpes. In recent years, morbidity of genital herpes has been rising heavely, and its impairment becomes severe increasingly. But now there are no workable medicine to control its infection and recurrence. It is crucial to develop vaccine to prevent genital herpes. Although immune responses induced by DNA vaccine are lower, DNA vaccine is the most favorable among the developed vaccines, for it is safe and simple to make. Furthermore, DNA vaccine can effectively stimulate cell-mediated and humoral immune responses, and can coexpress with cytokines to induce the most appropriate immune responses on the basis of need. Recently, foreign scholars have been working for enhancing immune responses of DNA vaccine by using molecular adjuvant, and had got some achievement. In order to construct effective DNA vaccine to prevent HSV-II infection and explore modulation mechanisms of immunity of molecular adjuvant, we have made the following works in this study
: 1. Construction of DNA Vaccine of Herpes Simplex Virus Type II gD:
According to HSV-II strain HG52 gD gene sequence in GenBank, a pair of primers were designed and the whole encoding sequence of HSV-II strain Sav gD
was amplified by PCR. The PCR product was inserted into pUCm-T vector. Subsequently it was digested by restriction endonucleases HindIII and XbaI, and inserted orientally into mammlian vector pcDNA3 at corresponding sites. Thus the recombinant was transformed into E.coli DH5 a . Identified by PCR, restriction enzymes analysis and sequencing, recombinant eukaryotic expression plasmid was named Pg. When Pg was transiently transfected into COS-7 cell, specific recombinant protein gD was demonstrated by in situ ELISA with specific gD McAb. It indicated that Pg was able to express in mammalian cells.
2. Construction of recombinant eukaryotic expression plasmid of murine chemokine MIP-1α
The total RNA was extracted from LPS-stimulated RAW264.7 cell before the whole encoding sequence of MIP-1 a was amplified by RT-PCR. The RT-PCR product was inserted into pUCm-T vector. After RT-PCR product was digested by restriction endonucleases Hindlll and Xbal in pUCm-T vector, it was inserted orientally into mammlian vector pcDNA3 at corresponding sites. Then the recombinant was transformed into E.coli DH5 a . Identified by PCR, restriction enzymes analysis and sequencing , recombinant eukaryotic expression plasmid was named Pm. Specific recombinant protein rMIP-1α transiently expressed in COS-7 cell was demonstrated by RT-PCR and boyden chemotaxis chamber. It suggested that Pm be able to express in mammalian cells.
3. Assessment for protective effects of Pg +Pm DNA vaccine on mice
Mice were injected in the quadriceps with 200ug Pg, Pg+Pm in a final volume of 200ul of NS separately. The control mice were immunized with pcDNA3 and NS, respectively. Immunization was made for three times. One month after the last immunization, mice were challenged intravaginally with 100LD50 of HSV-II strain
Sav. Mice were then examined daily to evaluate pathological conditions and survival rates within 14 days. The results showed that Pg+Pm DNA vaccine improved, the survival rate of mice and reduced both the number of mice with lesions and severity of herpetic lesions significantly. 4. Evaluation of the immune responses induced by Pg +Pm DNA vaccine:
To detect the specific antibody titers by ELISA and microneutralization test, we collected serum at the second week and the fouth week after the last immunization. Lymphocyte transformation test (LTT) was made using MTT in the first month after the last immunization. Pg+Pm DNA vaccine resulted in a moderate but not significant enhancement of gD-specific IgG levels and induced significant enhancement of Th-cell proliferation responses. These results illustrated that Pg+Pm DNA vaccine induced higher antigen-specific cell-mediated immune responses than Pg DNA vaccine alone.
In sum, we have successfully constructed the DNA vaccine of HSV-II strain Sav gD with MIP-1α
引文
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69．金冬雁，黎孟枫译．分子克隆实验指南[M]．第2版，北京：科学出版社．1996：19—22；322-323；793—797
70．颜子颖，王海林译．精编分子生物学实验指南．北京：科学出版社．1998：680—682．
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83．黄祯祥主编．医学病毒学基础及实验技术．北京：科学出版社．1990；142-148
84. Ghias HM, Nesbum AB, Kaiwar R, et al. Immunoselection of recombinant baculoviruses expressing high levels of biologically active herpes simplex virus type 1 glycoprotein D[J]. Arch Virol. 1991; 121:163
85．杜平主编．医学实验病毒学[M]．第一版．北京：人民军医出版社．1985；81-109
86. Stanberry LR, Cunningham AL, Mindel A, et al. Prospects for control of herpes simplex virus disease through immunization. Clin Infect Dis. 2000; 30(3): 549-566
87. Stanberry LR, Spruance SL, Cunningham AL, et al. Glycoprotein-D-adjuvant vaccine to prevent genital herpes. N Engl J Med. 2002; 347(21): 1652-1661
88. Nass PH, Elkins KL, Weir JP. Protective immunity against herpes simplex virus generated by DNA vaccination compared to natural infection. Vaccine. 2001; 19(11-12): 1538-1546
89. Morrison LA, Vaccines against genital herpes: progress and limitations. Drugs. 2002; 62(8): 1119-1129
90．孙树汉，戴建新，张平武等．核酸疫苗．第二军医大学出版社．2000；前言
91．Hsiung GD(许德芳节译)．生殖器疱疹：豚鼠模型的发病与化疗．国外医学·微生物学分册 1984；12：252-255
92. Bravo FJ, Stanberry LR, Kier AB, et al. Evalution of HPMPC therapy for primary and recurrent genital herpes in mice and guinea pigs. Antiviral Res. 1993; 21:59-72
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