人微小病毒B19-VP2基因真核表达质粒的构建
|
摘要
研究目的：人微小病毒B19(Human Parvovirus B19)系微小病毒属中唯一能致人类疾病的一种病原体，可引起传染性红斑、再障危象等多种人类疾病。目前尚来建立针对该病毒的特异性疫苗，对人微小病毒B19感染的预防和治疗是急待解决的问题。近年来国外开始把B19的衣壳蛋白作为疫苗抗原进行研究。本课题的目的是利用真核表达质粒pcDNA3.1为载体，将选择出的B19-VP2抗原表位丰富区基因片段重组其中，构建出pcDNA3.1-VP2重组表达载体，并将重组质粒免疫动物，观察其免疫效应，以探讨人微小病毒B19-VP2基因作为基因疫苗的可行性，为最终建立针对该病毒的特异性实用疫苗奠定基础。
方法：从人微小病毒B19的基因序列和结构分析中可获得B19-VP2的氨基酸序列，利用ANTHEWIN和DNASIS软件对B19-VP2的表位进行分析；在此基础上设计出B19-VP2靶区域引物，为了克隆方便和插入方向正确，特意在两条引物上分别加入HindⅢ和BamHⅠ内切酶位点；利用PCR技术从含B19病毒DNA序列的质粒中扩增出VP2基因；将其连接在pGEM-T质粒上，转化JM109，运用pGEM-T质粒的T7／SP6序列设计出引物对连接的正确性进行鉴定，利用AMP抗性对阳性克隆进行筛选，并进行DNA测序，以保证克隆的目的片段的正确性；将重组质粒pGEM-T-VP2和pcDNA3.1同时用Hind Ⅲ和BamH Ⅰ酶切，电泳纯化后进行亚克隆，转化细菌，再次筛选和鉴定出阳性克隆，并进行DNA测序。大量培养、提取、纯化pcDNA3.1-VP2，肌注家兔，动态采血，ELISA方法测特异性抗体。
结果:
1.表位分析:通过对VPZ的294一541位氨基酸序列分析，初步确定了9个表位。
它们分别是①VNSVSTKEGDSSNT②TGLSTGTS③SLRPGPVSQPYHHWDT④
工SHGQTTYGNAEDKEYQ⑤GRFPNEKEQ⑥MHTYFPNKGTQQYTD⑦FLKILPQSG⑧
FKLGPRKATGRWN⑨LYDPTATDAKQHHRHGYEK
2.靶基因扩增:以含有B19病毒全基因的质粒为模板，利用PCR扩增得到vPZ
基因中的靶基因(4168一4968)，共800个碱基对。
3.克隆和亚克隆重组子结果鉴定:靶基因与pGEMesT质粒连接后，以T7/SP6作为
引物对转化后的质粒进行筛选扩增，得到96ObP的阳性克隆，证实B19一VPZ靶基
因成功插入pG甜一T，即得到pGEM一T一VPZ重组质粒。将重组质粒pGEM，T一VPZ和
PcDNA3.l同时用Hindlll和B叨Hl酶切后进行亚克隆，以T7/SP6作为引物对转
化后的质粒进行扩增，得到 966bP的阳性克隆。证实B19一P2靶基因成功插入
peDNA3.1，即得到peDNA3.1一VPZ重组质粒。
4.克隆和亚克隆重组子测序结果:对鉴定得到的克隆和亚克隆重组子
PGEM-T一VPZ和PcDNA3.1一VPZ进行DNA测序，证实我们所克隆/亚克隆出的VPZ
基因片段的碱基组成与公开发表的vPZ序列完全一致，即成功构建pcDNA3.1一vPZ
真核表达载体。
5，ELISA结果:实验动物的体内产生了高效价的抗B19病毒的工gG抗体。
结论:本课题利用分子生物学软件分析出B19一VPZ抗原表位丰富区，确定了9
个抗原表位，主要集中于294一541位氨基酸序列中。利用分子生物学的方法，成
功构建含B19一VPZ基因的真核表达质粒PcDNA3.1一VPZ。并通过免疫接种，在动
物体内产生了较强的免疫应答。通过此项研究，为人微小病毒B19基因疫苗的研
制奠定了坚实的基础，并为进一步研究基于VPZ的表位生物学和表位疫苗提供了
详实的数据。
Objective: Human Parvovirus B19 is only kind of pathogen causing human disease, which may bring about such critical diseases as infectious erythema, aplastic anemia and so on, and yet no specific vaccine against the virus has been found out. .preventing and treating the infection of Human parvovirus B19 is urgent problem need to be solved. Recently recombination of B19 capsid protein as vaccine antigen is processing in fremdness. The objective of the project is using eukaryotic expression of plasmid pcDNA3.l as vector, selecting the gene fragment of abundance antigen epitope and recombinating it to vector, Constructing pcDNA3.l-VP2 eucaryotic expression of vector. Finally we injected the recombination plasmid directly into an experimental animal to observe its immune reaction in order to explore the feasibility of Human parvovirus B19-VP2 gene as genie vaccine. The subject lay a solid foundation for finally establishing specific utility vaccine of the virus.
Met hods: The gene sequence of B19-VP2 can be obtained from gene sequence and its structural analysis of Human parvovirus B19. The B19-VP2 target gene primer was designed on the basis of analysis of the epitope of B19-VP2 with software ANTHEWIN and DNASIS. In order to facilitate cloning and ensure the exactness of insert direction. We designedly joined Hindin and BamHI inscribed enzyme point into primer ;Gene sequence of VP2 was ampliticated from plasmid of DNA sequence of B19 virus with PCR method and was linked to pGEM-T plasmid, which was transformed into JM109. The primer designed with T7/sp6 sequence of pGEM-T plasmid was applied to identify the linking correctness. Furthermore; AMP fastness was also used to select the positive clone and to identify the sequence of DNA for the purpose of the correctness of objective fragment cloned. After the recombinated plasmid pGEM-T-VP2 and pcDNA 3.1 underwent enzyme cutting with both HindTTI and BamHI, and then with electrophoretic purification, they were linked and transformed into
bacteria. Once again the work was carried out on selecting and identifying positive clone and DNA sequence. Finally the specific antibody was identified with ELISA method after a series of working processes such as mass culturing, extracting and purifying pcDNA 3.1-VP2 and injecting it into a rabbit in tramuscularly and collecting its blood dynamically.
Resu 11: l.Epitope analysis: nine epitopes were primarily identified through analysis on
ISHGQTTYGNAEDKEYQ (5) GRFPNEKEQ (6) MHTYFPNKGTQQYTD (7) FLKILPQSG (8) FKLGPRKATGRWN(9)LYDPTATDAKQHHRHGYEK
2. Amplification of target gene : 800 base pairs were found out by way of whole gene plasmid of B19 virus as template and target gene (4168-4968) in gene VP2 amplificated by PCR.
3.Result of recombination and identification of clone and subclone: the positive clone of 960bp was obtained through linking between target gene and pGEM-T plasmid, and then amplificating transformed plasmid by primer T7/sp6.that proved B19-VP2 target gene insert pGEM-T successfully, and we gained recombined pGEM-T-VP2 plasmid. The positive clone 966bp was obtained by means of Linking recombinated plasmid pGEM-T-VP2 and pcDNA3.l after undergoing enzyme cutting with both Hindm and Bam HI. And by means of amplification of plasmid transformed by primer t7/sp6. .that proved B19-VP2 target gene insert pcDNA3.l successfully and we gained recombined pcDNA3.l-VP2 plasmid.
4. .Result of sequence analysis of clone and subclone: The identification of DNA in positive clone pGEM-T-VP2 and pcDNA3.l-VP2 demonstrated that the base pains of our cloned or subcloned VP2 gene fragments were identical with the VP2 sequence published in the world .We Constructed Eucaryotic Expression of vector pcDNA3. 1-VP2 successfully.
5.Result of ELISA: Highly concentration IgG against B19 virus was generated in the rabbit.
Conclusion: This subject make use of molecular biology software analyzing the abundance antigen epitope region of B19-VP2, nine epitopes were concentrated primarily on No.294-541 of vp2.. with molecular biological
方法：从人微小病毒B19的基因序列和结构分析中可获得B19-VP2的氨基酸序列，利用ANTHEWIN和DNASIS软件对B19-VP2的表位进行分析；在此基础上设计出B19-VP2靶区域引物，为了克隆方便和插入方向正确，特意在两条引物上分别加入HindⅢ和BamHⅠ内切酶位点；利用PCR技术从含B19病毒DNA序列的质粒中扩增出VP2基因；将其连接在pGEM-T质粒上，转化JM109，运用pGEM-T质粒的T7／SP6序列设计出引物对连接的正确性进行鉴定，利用AMP抗性对阳性克隆进行筛选，并进行DNA测序，以保证克隆的目的片段的正确性；将重组质粒pGEM-T-VP2和pcDNA3.1同时用Hind Ⅲ和BamH Ⅰ酶切，电泳纯化后进行亚克隆，转化细菌，再次筛选和鉴定出阳性克隆，并进行DNA测序。大量培养、提取、纯化pcDNA3.1-VP2，肌注家兔，动态采血，ELISA方法测特异性抗体。
结果:
1.表位分析:通过对VPZ的294一541位氨基酸序列分析，初步确定了9个表位。
它们分别是①VNSVSTKEGDSSNT②TGLSTGTS③SLRPGPVSQPYHHWDT④
工SHGQTTYGNAEDKEYQ⑤GRFPNEKEQ⑥MHTYFPNKGTQQYTD⑦FLKILPQSG⑧
FKLGPRKATGRWN⑨LYDPTATDAKQHHRHGYEK
2.靶基因扩增:以含有B19病毒全基因的质粒为模板，利用PCR扩增得到vPZ
基因中的靶基因(4168一4968)，共800个碱基对。
3.克隆和亚克隆重组子结果鉴定:靶基因与pGEMesT质粒连接后，以T7/SP6作为
引物对转化后的质粒进行筛选扩增，得到96ObP的阳性克隆，证实B19一VPZ靶基
因成功插入pG甜一T，即得到pGEM一T一VPZ重组质粒。将重组质粒pGEM，T一VPZ和
PcDNA3.l同时用Hindlll和B叨Hl酶切后进行亚克隆，以T7/SP6作为引物对转
化后的质粒进行扩增，得到 966bP的阳性克隆。证实B19一P2靶基因成功插入
peDNA3.1，即得到peDNA3.1一VPZ重组质粒。
4.克隆和亚克隆重组子测序结果:对鉴定得到的克隆和亚克隆重组子
PGEM-T一VPZ和PcDNA3.1一VPZ进行DNA测序，证实我们所克隆/亚克隆出的VPZ
基因片段的碱基组成与公开发表的vPZ序列完全一致，即成功构建pcDNA3.1一vPZ
真核表达载体。
5，ELISA结果:实验动物的体内产生了高效价的抗B19病毒的工gG抗体。
结论:本课题利用分子生物学软件分析出B19一VPZ抗原表位丰富区，确定了9
个抗原表位，主要集中于294一541位氨基酸序列中。利用分子生物学的方法，成
功构建含B19一VPZ基因的真核表达质粒PcDNA3.1一VPZ。并通过免疫接种，在动
物体内产生了较强的免疫应答。通过此项研究，为人微小病毒B19基因疫苗的研
制奠定了坚实的基础，并为进一步研究基于VPZ的表位生物学和表位疫苗提供了
详实的数据。
Objective: Human Parvovirus B19 is only kind of pathogen causing human disease, which may bring about such critical diseases as infectious erythema, aplastic anemia and so on, and yet no specific vaccine against the virus has been found out. .preventing and treating the infection of Human parvovirus B19 is urgent problem need to be solved. Recently recombination of B19 capsid protein as vaccine antigen is processing in fremdness. The objective of the project is using eukaryotic expression of plasmid pcDNA3.l as vector, selecting the gene fragment of abundance antigen epitope and recombinating it to vector, Constructing pcDNA3.l-VP2 eucaryotic expression of vector. Finally we injected the recombination plasmid directly into an experimental animal to observe its immune reaction in order to explore the feasibility of Human parvovirus B19-VP2 gene as genie vaccine. The subject lay a solid foundation for finally establishing specific utility vaccine of the virus.
Met hods: The gene sequence of B19-VP2 can be obtained from gene sequence and its structural analysis of Human parvovirus B19. The B19-VP2 target gene primer was designed on the basis of analysis of the epitope of B19-VP2 with software ANTHEWIN and DNASIS. In order to facilitate cloning and ensure the exactness of insert direction. We designedly joined Hindin and BamHI inscribed enzyme point into primer ;Gene sequence of VP2 was ampliticated from plasmid of DNA sequence of B19 virus with PCR method and was linked to pGEM-T plasmid, which was transformed into JM109. The primer designed with T7/sp6 sequence of pGEM-T plasmid was applied to identify the linking correctness. Furthermore; AMP fastness was also used to select the positive clone and to identify the sequence of DNA for the purpose of the correctness of objective fragment cloned. After the recombinated plasmid pGEM-T-VP2 and pcDNA 3.1 underwent enzyme cutting with both HindTTI and BamHI, and then with electrophoretic purification, they were linked and transformed into
bacteria. Once again the work was carried out on selecting and identifying positive clone and DNA sequence. Finally the specific antibody was identified with ELISA method after a series of working processes such as mass culturing, extracting and purifying pcDNA 3.1-VP2 and injecting it into a rabbit in tramuscularly and collecting its blood dynamically.
Resu 11: l.Epitope analysis: nine epitopes were primarily identified through analysis on
ISHGQTTYGNAEDKEYQ (5) GRFPNEKEQ (6) MHTYFPNKGTQQYTD (7) FLKILPQSG (8) FKLGPRKATGRWN(9)LYDPTATDAKQHHRHGYEK
2. Amplification of target gene : 800 base pairs were found out by way of whole gene plasmid of B19 virus as template and target gene (4168-4968) in gene VP2 amplificated by PCR.
3.Result of recombination and identification of clone and subclone: the positive clone of 960bp was obtained through linking between target gene and pGEM-T plasmid, and then amplificating transformed plasmid by primer T7/sp6.that proved B19-VP2 target gene insert pGEM-T successfully, and we gained recombined pGEM-T-VP2 plasmid. The positive clone 966bp was obtained by means of Linking recombinated plasmid pGEM-T-VP2 and pcDNA3.l after undergoing enzyme cutting with both Hindm and Bam HI. And by means of amplification of plasmid transformed by primer t7/sp6. .that proved B19-VP2 target gene insert pcDNA3.l successfully and we gained recombined pcDNA3.l-VP2 plasmid.
4. .Result of sequence analysis of clone and subclone: The identification of DNA in positive clone pGEM-T-VP2 and pcDNA3.l-VP2 demonstrated that the base pains of our cloned or subcloned VP2 gene fragments were identical with the VP2 sequence published in the world .We Constructed Eucaryotic Expression of vector pcDNA3. 1-VP2 successfully.
5.Result of ELISA: Highly concentration IgG against B19 virus was generated in the rabbit.
Conclusion: This subject make use of molecular biology software analyzing the abundance antigen epitope region of B19-VP2, nine epitopes were concentrated primarily on No.294-541 of vp2.. with molecular biological
引文
1 杨月．基因疫苗．中国临床医生，1999，27(11)：59～59
2 姬新颖，周建军，王海涛等．基因疫苗的特点、应用和最新进展．微生物学免疫学进展，1999,27(1)：64～67
3 聂青和，李梦东．某些传染病基因疫苗的研究进展．中华传染病杂志,1998，16(4)：247～249
4 Facio VM. "Naked DNA" transfer technology for genetic vaccination against infectious diseases. Res Virol, 1997,148:101～108
5 Ransay AJ, Ramshaw IA, Ada GL.DNA immunization.Immunol.Cell Bio1,1997,75(4):360～363
6 Yokoyanna M,Hasseitt DE,Zhang J,et al.DNA immunizatiobn can stimulate forid local inflamation, and the antiviral immunity induced variesdependingon injection site.Vaccine, 1997,15 (5):553～560
7 张友金，陈其龙，陶志华，等．乙肝基因疫苗免疫效果观察．浙江预防医到学，2000，12(1)：11～12
8 秦山，唐红，等．乙肝基因疫苗系列质粒的构建及其在真核细胞中的表达．华西医大学报，2001，32(2)：172～174
9 谭德明，刘双虎，等．乙型肝炎病毒全S基因疫苗与HBsAg基因疫苗诱导小鼠的特异性免疫应答比较．中华传染病杂志,2001，19(4)：240～241
10 王勇．肿瘤基因疫苗——肿瘤免疫治疗的新途径．国外医学肿瘤学分册，2000，27(3)：129～132
11 Ransay AJ, Ramshaw IA, Ada GL. DNA immunization. Immunol Cell Bio1,1997,75(4):360～363
12 吴晓羽，赵铁强，等．柯萨奇病毒B1/B3型二价VP1基因疫苗的构建及其免疫试验．中华医学杂志，2001，81(8)：480～484
13 Chia j k s,Jackson B.Myopericarditis due to parvovirus B19 in an adult.Clin infect dis,1996,23:200～201
14 Brown K E,Young N S.Parvovirus B19 in human disease.Annu Rev Med,1997,48:59～67
15 成胜权，张国成，许东亮．幼年类风湿性关节炎与人细小病毒B19感染的关系及其临床特征．中华儿科杂志，2000，38(8)：477～479
16 Flunker G,Peters A,Wiersbitzky Setal. Persistent parvovirus B19 infection in immunocompromised children[J]. Med Microbiol Immunol Berl, 1998,186(4): 189～194
17 漆洪波．妊娠期人细小病毒B19感染．实用妇产料杂志,1998,14(1)：7～8．
18 Kinney JS,Anderson LJ,Farrar J, et al.Risk of adverse outcomes of pregnancy after human parvovirus B19 infection.J infect Dis,1998,157:663
19 王晓明．人微小病毒B19致人类感染性疾病的研究进展．国外医学妇幼保健分册，1998，9(2)：73～77
20 王晓明，张国成，韩美玉，等．先天性心脏病心脏组织中B19-DNA的检测研究．中华儿科杂志，1998,36(6)：364～365
21 江凌晓．人微小病毒B19分子生物学研究进展届外医学病毒学分册，2000，7(1)：11～14
22 孙新．人细小病毒B19基因变异研究进展．国外医学流行病传染病学分册，1999，26(2)：73～76
23 William A Beard, Samuel H,Wilson.Structural design of eukaryotie DNA repair polymerase,DNA polymerase β, mutation Res,2000,460:231
24 L.J.Deterding,R.Prasad, S.H.Wilson, et al.Tomer, Ide dification of the Sehiff base nucleophile in the DNA polymerase dRP lyase mechanism. J.Biol.Chem,2000,275:10463
25 J.K.Horton,R.Prasad,E.Hou, et al.Proteetion against methylationinduced cytotxicity by DNA polymerase beta depended long patch base excision repair.J.Biol.Chem,2000,275:4460
26 M.W.Maciejewski, D. Liu, R. Prasad, et al. Backbone dynamics and refined solution structure of the N-terminal domain of DNA polymerase β. Correlation with DNA binding and dRP lyase activity. Mol. Biol. 2000, 296:229
2 姬新颖，周建军，王海涛等．基因疫苗的特点、应用和最新进展．微生物学免疫学进展，1999,27(1)：64～67
3 聂青和，李梦东．某些传染病基因疫苗的研究进展．中华传染病杂志,1998，16(4)：247～249
4 Facio VM. "Naked DNA" transfer technology for genetic vaccination against infectious diseases. Res Virol, 1997,148:101～108
5 Ransay AJ, Ramshaw IA, Ada GL.DNA immunization.Immunol.Cell Bio1,1997,75(4):360～363
6 Yokoyanna M,Hasseitt DE,Zhang J,et al.DNA immunizatiobn can stimulate forid local inflamation, and the antiviral immunity induced variesdependingon injection site.Vaccine, 1997,15 (5):553～560
7 张友金，陈其龙，陶志华，等．乙肝基因疫苗免疫效果观察．浙江预防医到学，2000，12(1)：11～12
8 秦山，唐红，等．乙肝基因疫苗系列质粒的构建及其在真核细胞中的表达．华西医大学报，2001，32(2)：172～174
9 谭德明，刘双虎，等．乙型肝炎病毒全S基因疫苗与HBsAg基因疫苗诱导小鼠的特异性免疫应答比较．中华传染病杂志,2001，19(4)：240～241
10 王勇．肿瘤基因疫苗——肿瘤免疫治疗的新途径．国外医学肿瘤学分册，2000，27(3)：129～132
11 Ransay AJ, Ramshaw IA, Ada GL. DNA immunization. Immunol Cell Bio1,1997,75(4):360～363
12 吴晓羽，赵铁强，等．柯萨奇病毒B1/B3型二价VP1基因疫苗的构建及其免疫试验．中华医学杂志，2001，81(8)：480～484
13 Chia j k s,Jackson B.Myopericarditis due to parvovirus B19 in an adult.Clin infect dis,1996,23:200～201
14 Brown K E,Young N S.Parvovirus B19 in human disease.Annu Rev Med,1997,48:59～67
15 成胜权，张国成，许东亮．幼年类风湿性关节炎与人细小病毒B19感染的关系及其临床特征．中华儿科杂志，2000，38(8)：477～479
16 Flunker G,Peters A,Wiersbitzky Setal. Persistent parvovirus B19 infection in immunocompromised children[J]. Med Microbiol Immunol Berl, 1998,186(4): 189～194
17 漆洪波．妊娠期人细小病毒B19感染．实用妇产料杂志,1998,14(1)：7～8．
18 Kinney JS,Anderson LJ,Farrar J, et al.Risk of adverse outcomes of pregnancy after human parvovirus B19 infection.J infect Dis,1998,157:663
19 王晓明．人微小病毒B19致人类感染性疾病的研究进展．国外医学妇幼保健分册，1998，9(2)：73～77
20 王晓明，张国成，韩美玉，等．先天性心脏病心脏组织中B19-DNA的检测研究．中华儿科杂志，1998,36(6)：364～365
21 江凌晓．人微小病毒B19分子生物学研究进展届外医学病毒学分册，2000，7(1)：11～14
22 孙新．人细小病毒B19基因变异研究进展．国外医学流行病传染病学分册，1999，26(2)：73～76
23 William A Beard, Samuel H,Wilson.Structural design of eukaryotie DNA repair polymerase,DNA polymerase β, mutation Res,2000,460:231
24 L.J.Deterding,R.Prasad, S.H.Wilson, et al.Tomer, Ide dification of the Sehiff base nucleophile in the DNA polymerase dRP lyase mechanism. J.Biol.Chem,2000,275:10463
25 J.K.Horton,R.Prasad,E.Hou, et al.Proteetion against methylationinduced cytotxicity by DNA polymerase beta depended long patch base excision repair.J.Biol.Chem,2000,275:4460
26 M.W.Maciejewski, D. Liu, R. Prasad, et al. Backbone dynamics and refined solution structure of the N-terminal domain of DNA polymerase β. Correlation with DNA binding and dRP lyase activity. Mol. Biol. 2000, 296:229