pcDNA3/hIL-22和pcDNA3/hIL-22-VP1质粒的构建及其免疫作用研究
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摘要
目的：人白细胞介素-22（human interleukin-22，hIL-22）是2000年由Dumoutier等新发现的一种细胞因子，位于人染色体12q15，成熟的hIL-22是由179个氨基酸组成，与人白细胞介素有23%的同源性；主要由T淋巴细胞产生，包括CD4(+) T细胞、CD45RA(+) T细胞和CD45RO(+) T细胞，在NK细胞中也可微量产生；hIL-22通过其受体起作用，hIL-22与受体结合可以激活STAT1、3、5信号传导系统、激活JAK1和TYK2系统和激活MEK-ERK-RSK、JNK/SPAK和P38激酶等三个MAPK路径，由此介导基因的转录和翻译。它的主要生物学活性有：使肝细胞在急性期反应中急性期产物上调；在胰腺的炎症反应中可诱导胰腺炎相关蛋白PAP1的表达；另外发现它与淋巴瘤的诱导、哮喘易感性、抗寄生虫感染免疫、和B1淋巴细胞扩张等有关；在免疫调节方面，hIL-22可抑制Th2细胞产生IL-4，而对Th1细胞IFNγ产生无影响；Wolk等将hIL-22归类于Th1型细胞因子，认为它具有介导细胞免疫应答的作用。其生物学作用，还有待于进一步深入研究。而白细胞介素22的克隆及真核表达载体的构建，就成为了这方面工作的一个基础。据此，我们克隆了IL-22基因，并构建了PCDNA3/IL-22真核表达质粒。观察了其对BalB/C小鼠血
清抗体产生的影响及CVB3病毒攻击的保护作用。
柯萨奇病毒B组（Coxsackievirus group B, CVB）属小RNA病毒科肠道病毒属，是引起人类病毒性心肌炎的主要病原体。临床上至少有50%的急性心肌炎和大约25%大扩张性心肌病是由该病毒引起，其中以CVB3为最常见。目前尚无有效的CVB3灭活疫苗和减毒活疫苗应用于临床，而基因疫苗则为防治CVB3提供了可能性。CVB3编码VP1蛋白的基因较为保守，VP1蛋白可刺激机体产生具有保护作用的中和抗体。目前国内外多以CVB3的VP1基因构建的真核表达质粒系统作为CVB3的基因疫苗。但从已有的研究显示，以单一的VP1基因构建的基因疫苗，虽然可以诱导机体产生抗体，但因抗体效价不高而无明显保护作用。如何增强VP1基因的免疫原性，提高抗体的保护作用，是目前亟需解决的问题。在DNA疫苗中加入细胞因子，免疫共刺激分子、免疫刺激序列或免疫调节分子，作为分子佐剂来提高DNA疫苗的免疫作用，可能是解决此问题的一条途径。而IL-22作为细胞因子对VP1基因疫苗有无增强作用，是一个值得探讨的问题。据此，我们构建了hIL-22和柯萨奇病毒VP1基因的双重表达质粒，观察了BalB/C小鼠接种该质粒后血清抗体水平产生的情况及其对CVB攻击的保护作用
方法：（1）从人新鲜外周血分离淋巴细胞，进行淋巴细胞培养，在刀豆蛋白Ａ刺激后，提取总RNA，用自行设计的人hIL-22特异性引物P57(+)5'-GAAGGATCCGTTATGGCCGCCCTG CAGAA-3'、P596(-)5′GGACTCGAGTCAAATGCAGGCATTTCTCAG3′进行RT-PCR扩
增；（2）将扩增产物与pGEM-T 载体连接，转化DH5a 大肠杆菌，筛选重组子，提取质粒进行酶切鉴定，将酶切鉴定结果阳性的菌株送检测序；（3）将pGEM-T-hIL-22进行双酶切(BamHⅠ和XhoⅠ)，与经过同样两种酶切的pcDNA3载体连接，经过转化、筛选和酶切鉴定后，构建成真核表达重组质粒pcDNA3-hIL-22。（4）以pcDNA3-VP1质粒为模板用自行设计的引物P209(+)5'-GCATAGATCTGCGATGTACGGGCCAGATAT-3'和P1249(-)5'-ATACAA T TGTCCCCAGCATGCCTGCTAT3'，进行PCR反应，扩增出带有CMV启动子和BGH加尾信号等完整真核表达构件的VP1基因（CMVp-VP1-BGHp）；（5）将CMVp-VP1-BGHp双酶切（BglII和NruⅠ），与同样双酶切的pcDNA3-IL-22质粒进行连接，经过转化、筛选和鉴定，构建成双表达真核重组质粒pcDNA3-VP1/IL-22；用碱裂解法进行质粒大制备，以获得足够的质粒DNA接种小鼠；（4）将100只4-6周龄的BALB/c小鼠随机分为5组，分别为：盐水对照组、pcDNA3-hIL-22对照组、pcDNA3-VP1对照组、pcDNA3-VP1和pcDNA3-hIL-22混和质粒对照组、pcDNA3-VP1-hIL-22组，采用肌肉注射接种质粒方法，除盐水对照组每只小鼠注射0.2ml盐水外，其他各组每只小鼠注射100ug相应质粒，每隔2周注射一次，共注射3次。每次免疫后的第十三天，断尾取血检测CVB3中和抗体。3 次免疫后用1000TCID50的CVB3病毒感染小鼠，观察各组小鼠死亡情况。（5）使用秩和检验中的Kruskal-Wallis法，分别对每次免疫后各组小鼠产生的中和抗体效价和每组小鼠三次免疫所产生的中和抗体效价进行多组比较；使
用秩和检验的Nemenyi法对每次免疫后各组小鼠之间进行两两比较；使用Log-Rank秩检验法进行各组小鼠生存率的两两比较。
结果:（1）外周血淋巴细胞在培养过程中，生长状况良好，并在Con A刺激后淋巴细胞明显增大、明亮。经RT-PCR所得到的基因片段与预期大小一致（560bp），与pGEM T连接后，酶切初步鉴定结果与预期相符，经过测序后，证实所得基因序列与人白细胞介素22（hIL-22）基因序列完全一致(57-596bp)。（2）hIL-22与pcDNA3连接后，酶切鉴定结果与预期相符，表明hIL-22的真核表达质粒构建成功。（3）hIL-22与VP1基因在双表达质粒构建过程中，经过连接、转化、筛选后，酶切鉴定结果与预期相符，再经过测序证实，构建的pcDNA3-VP1-hIL-22重组质粒目的基因与CVB3-VP1和IL-22的标准序列相同，证明pcDNA
Objective: Human interlerkin-22 is a novel cytokine discovered by Dumoutier et al. in 2000. Mature hIL-22 is constituted of 179 amino acids, and has 23% homogeneity with interleukin-10; It is produced mainly by T lymphocyte, including CD4(+) T lymphocyte, and NK cells can produce little of it. By binding with its receptor, hIL-22 could activate STAT1、3、5 signaling pathways. JAK1 TYK2 and MEK-ERK-RSK、JNK/SPAK and P38 MAPK Signaling pathways, that medicate the transcription and translation of genes. Its main biological effect including: up-regulating the expression of acute reactive proteins in liver cells; inducing the expression of pancreatitis associated Protein PAP1 in pancreatitis. In addition, it was found to be related to lymphoma inducing. asthma susceptibility , anti-parasite immune response and cell expansion of B1 lymphocyte; Regarding Immunological regulation, hIL-22 inhibits the production of IL-4 by Th2 lymhpocytes but has no influence on the production of IFN-γin these cells. Wolk et al. attributed IL-22 to Th1 type cytokine and believed it could mediate cell immune response. More biological functions of IL-22 remain to be investigated, and the construction of
eukaryotic expression plasmid of hIL-22 will lay a foundation for this. Thus, we cloned the gene of hIL-22, constructed the plasmid of pcDNA3/hIL-22 and observed its protective effect on CVB3 challenged BALB/c mice. Coxsackievirus belong to picornavirus, and coxsackievirus group B (CVB) is the prodominant pathogen of human viral myocarditis. Clinically, at least 50% acute myocarditis are caused by this virus, with CVB3 being the most common. Until now, there is no effective CVB3 vaccine available and DNA vaccine provide an alternative for treatment and prevention of CVB infections. The gene of CVB3 VP1 protein is a conservative gene, and the protein that encoded can stimulate the production of antibody. At present, most investigators have constructed the eukaryotic expressing plasmid of CVB3 VP1 gene to use as a gene vaccine. But the ported observations suggested that although the vaccine constructed with VP1 alone could induce the production of antibody, the titers of antibody produced were usually too low to provide protection. Therefore, enhancing the protective effect of VP1 gene vaccine is so far a problem need to be resolved. Adding genes of cytokine, costimulatory factor, ISS or immunological regulatory molecular as a molecular adjuvant, to the vaccine seems to be a possible way to solve this problem. As a cytokine, whether the IL-22 has or not the immune enhancing effect is worthy to investigate. Thus, we constructed a double expressing plasmid of pcDNA3/hIL-22-VP1 and observed its effect on the
production of antibody and protective effect against CVB3 infection in CVB3 challenged mice.
Methods: ⑴ Lymphocytes were isolated from human peripheral blood and the RNA was extracted after stimulating with ConA; PT-PCR was performed using a pair of primers specific to hIL-22, P57(+)5'-GAAGGATCCGTTATGGCCGCCCTG CAGAA-3‘、P596(-)5′GGACTCGAGTCAAATGCAGGCATTTCTCAG
-3′,to amplifying the gene fragment of hIL-22, ⑵ The amplified product was linked to the plasmid pGEMT and competent DH5αwas transformed. The plasmid DNA was extracted and identified by enzymes digesting and sequencing. ⑶ The pGEM-T/hIL-22 was cut by BamHⅠand XhoⅠ, and linked to pcDNA3 that had be cut using the same enzymes to construct a eukaryotic expressing plasmid pcDNA3/hIL-22. After transformation, the recombined plasmid was identified by enzymes cutting. ⑷ Using the plasmid pcDNA3/VP1 as a plate, PCR was performed to amplifying a VP1 gene fragment that carried the genes fragments (CMVp-VP1-BGHp) of CMV promotor and BGH Ploy A tailing Signal. The primers used were P209(+)5'-GCATAGATCTGCGATGTACGGGCCAGATAT-3'和P1249 (-)5'-ATACAATTGTCCCCAGCATGCCTGCTAT-3'⑸ The amplified PCR product CMV-VP1-BGHp was cut by BglⅡand NruⅠand linked to pcDNA3/IL-22 that had be cut by the same Enzymes, to construct the eukaryotic double expressing plasmid pcDNA3/IL-22-V
清抗体产生的影响及CVB3病毒攻击的保护作用。
柯萨奇病毒B组（Coxsackievirus group B, CVB）属小RNA病毒科肠道病毒属，是引起人类病毒性心肌炎的主要病原体。临床上至少有50%的急性心肌炎和大约25%大扩张性心肌病是由该病毒引起，其中以CVB3为最常见。目前尚无有效的CVB3灭活疫苗和减毒活疫苗应用于临床，而基因疫苗则为防治CVB3提供了可能性。CVB3编码VP1蛋白的基因较为保守，VP1蛋白可刺激机体产生具有保护作用的中和抗体。目前国内外多以CVB3的VP1基因构建的真核表达质粒系统作为CVB3的基因疫苗。但从已有的研究显示，以单一的VP1基因构建的基因疫苗，虽然可以诱导机体产生抗体，但因抗体效价不高而无明显保护作用。如何增强VP1基因的免疫原性，提高抗体的保护作用，是目前亟需解决的问题。在DNA疫苗中加入细胞因子，免疫共刺激分子、免疫刺激序列或免疫调节分子，作为分子佐剂来提高DNA疫苗的免疫作用，可能是解决此问题的一条途径。而IL-22作为细胞因子对VP1基因疫苗有无增强作用，是一个值得探讨的问题。据此，我们构建了hIL-22和柯萨奇病毒VP1基因的双重表达质粒，观察了BalB/C小鼠接种该质粒后血清抗体水平产生的情况及其对CVB攻击的保护作用
方法：（1）从人新鲜外周血分离淋巴细胞，进行淋巴细胞培养，在刀豆蛋白Ａ刺激后，提取总RNA，用自行设计的人hIL-22特异性引物P57(+)5'-GAAGGATCCGTTATGGCCGCCCTG CAGAA-3'、P596(-)5′GGACTCGAGTCAAATGCAGGCATTTCTCAG3′进行RT-PCR扩
增；（2）将扩增产物与pGEM-T 载体连接，转化DH5a 大肠杆菌，筛选重组子，提取质粒进行酶切鉴定，将酶切鉴定结果阳性的菌株送检测序；（3）将pGEM-T-hIL-22进行双酶切(BamHⅠ和XhoⅠ)，与经过同样两种酶切的pcDNA3载体连接，经过转化、筛选和酶切鉴定后，构建成真核表达重组质粒pcDNA3-hIL-22。（4）以pcDNA3-VP1质粒为模板用自行设计的引物P209(+)5'-GCATAGATCTGCGATGTACGGGCCAGATAT-3'和P1249(-)5'-ATACAA T TGTCCCCAGCATGCCTGCTAT3'，进行PCR反应，扩增出带有CMV启动子和BGH加尾信号等完整真核表达构件的VP1基因（CMVp-VP1-BGHp）；（5）将CMVp-VP1-BGHp双酶切（BglII和NruⅠ），与同样双酶切的pcDNA3-IL-22质粒进行连接，经过转化、筛选和鉴定，构建成双表达真核重组质粒pcDNA3-VP1/IL-22；用碱裂解法进行质粒大制备，以获得足够的质粒DNA接种小鼠；（4）将100只4-6周龄的BALB/c小鼠随机分为5组，分别为：盐水对照组、pcDNA3-hIL-22对照组、pcDNA3-VP1对照组、pcDNA3-VP1和pcDNA3-hIL-22混和质粒对照组、pcDNA3-VP1-hIL-22组，采用肌肉注射接种质粒方法，除盐水对照组每只小鼠注射0.2ml盐水外，其他各组每只小鼠注射100ug相应质粒，每隔2周注射一次，共注射3次。每次免疫后的第十三天，断尾取血检测CVB3中和抗体。3 次免疫后用1000TCID50的CVB3病毒感染小鼠，观察各组小鼠死亡情况。（5）使用秩和检验中的Kruskal-Wallis法，分别对每次免疫后各组小鼠产生的中和抗体效价和每组小鼠三次免疫所产生的中和抗体效价进行多组比较；使
用秩和检验的Nemenyi法对每次免疫后各组小鼠之间进行两两比较；使用Log-Rank秩检验法进行各组小鼠生存率的两两比较。
结果:（1）外周血淋巴细胞在培养过程中，生长状况良好，并在Con A刺激后淋巴细胞明显增大、明亮。经RT-PCR所得到的基因片段与预期大小一致（560bp），与pGEM T连接后，酶切初步鉴定结果与预期相符，经过测序后，证实所得基因序列与人白细胞介素22（hIL-22）基因序列完全一致(57-596bp)。（2）hIL-22与pcDNA3连接后，酶切鉴定结果与预期相符，表明hIL-22的真核表达质粒构建成功。（3）hIL-22与VP1基因在双表达质粒构建过程中，经过连接、转化、筛选后，酶切鉴定结果与预期相符，再经过测序证实，构建的pcDNA3-VP1-hIL-22重组质粒目的基因与CVB3-VP1和IL-22的标准序列相同，证明pcDNA
Objective: Human interlerkin-22 is a novel cytokine discovered by Dumoutier et al. in 2000. Mature hIL-22 is constituted of 179 amino acids, and has 23% homogeneity with interleukin-10; It is produced mainly by T lymphocyte, including CD4(+) T lymphocyte, and NK cells can produce little of it. By binding with its receptor, hIL-22 could activate STAT1、3、5 signaling pathways. JAK1 TYK2 and MEK-ERK-RSK、JNK/SPAK and P38 MAPK Signaling pathways, that medicate the transcription and translation of genes. Its main biological effect including: up-regulating the expression of acute reactive proteins in liver cells; inducing the expression of pancreatitis associated Protein PAP1 in pancreatitis. In addition, it was found to be related to lymphoma inducing. asthma susceptibility , anti-parasite immune response and cell expansion of B1 lymphocyte; Regarding Immunological regulation, hIL-22 inhibits the production of IL-4 by Th2 lymhpocytes but has no influence on the production of IFN-γin these cells. Wolk et al. attributed IL-22 to Th1 type cytokine and believed it could mediate cell immune response. More biological functions of IL-22 remain to be investigated, and the construction of
eukaryotic expression plasmid of hIL-22 will lay a foundation for this. Thus, we cloned the gene of hIL-22, constructed the plasmid of pcDNA3/hIL-22 and observed its protective effect on CVB3 challenged BALB/c mice. Coxsackievirus belong to picornavirus, and coxsackievirus group B (CVB) is the prodominant pathogen of human viral myocarditis. Clinically, at least 50% acute myocarditis are caused by this virus, with CVB3 being the most common. Until now, there is no effective CVB3 vaccine available and DNA vaccine provide an alternative for treatment and prevention of CVB infections. The gene of CVB3 VP1 protein is a conservative gene, and the protein that encoded can stimulate the production of antibody. At present, most investigators have constructed the eukaryotic expressing plasmid of CVB3 VP1 gene to use as a gene vaccine. But the ported observations suggested that although the vaccine constructed with VP1 alone could induce the production of antibody, the titers of antibody produced were usually too low to provide protection. Therefore, enhancing the protective effect of VP1 gene vaccine is so far a problem need to be resolved. Adding genes of cytokine, costimulatory factor, ISS or immunological regulatory molecular as a molecular adjuvant, to the vaccine seems to be a possible way to solve this problem. As a cytokine, whether the IL-22 has or not the immune enhancing effect is worthy to investigate. Thus, we constructed a double expressing plasmid of pcDNA3/hIL-22-VP1 and observed its effect on the
production of antibody and protective effect against CVB3 infection in CVB3 challenged mice.
Methods: ⑴ Lymphocytes were isolated from human peripheral blood and the RNA was extracted after stimulating with ConA; PT-PCR was performed using a pair of primers specific to hIL-22, P57(+)5'-GAAGGATCCGTTATGGCCGCCCTG CAGAA-3‘、P596(-)5′GGACTCGAGTCAAATGCAGGCATTTCTCAG
-3′,to amplifying the gene fragment of hIL-22, ⑵ The amplified product was linked to the plasmid pGEMT and competent DH5αwas transformed. The plasmid DNA was extracted and identified by enzymes digesting and sequencing. ⑶ The pGEM-T/hIL-22 was cut by BamHⅠand XhoⅠ, and linked to pcDNA3 that had be cut using the same enzymes to construct a eukaryotic expressing plasmid pcDNA3/hIL-22. After transformation, the recombined plasmid was identified by enzymes cutting. ⑷ Using the plasmid pcDNA3/VP1 as a plate, PCR was performed to amplifying a VP1 gene fragment that carried the genes fragments (CMVp-VP1-BGHp) of CMV promotor and BGH Ploy A tailing Signal. The primers used were P209(+)5'-GCATAGATCTGCGATGTACGGGCCAGATAT-3'和P1249 (-)5'-ATACAATTGTCCCCAGCATGCCTGCTAT-3'⑸ The amplified PCR product CMV-VP1-BGHp was cut by BglⅡand NruⅠand linked to pcDNA3/IL-22 that had be cut by the same Enzymes, to construct the eukaryotic double expressing plasmid pcDNA3/IL-22-V
引文
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20 receptor complex.J Bio Chem 2001, 276(4): 2575~2732.
21 Lejeune D,Dumoutier L,Constantinescu S,et al. Interleukin-22(IL-22) activates the JAK/STAT,ERK, JNK, and p38 map kinase pathways in a rat hepatoma cell line. J Bio Chem,2002,277(37):33676~33682.
22 Sato Y,Roman H,Tighe H,et al.Immunostimalatory DNA sequences necessary for effective inuadermal gene immunization.science,1996,273(3):352~354
23 Klinman DM, Yi AK,Krieg AM,et al.Cpg modifs presont in b-acteriul DNA rapidly induce lymphoeytes to secrete interleukin 6,interleukin 12 and interferon gamma.Pro DNA Soi USA,1996,93(7):2879~2883.
24 Pisetsky DS.DNA and the immune system.Ann Intern Med,1997,126(2):169~171.
25 Thalhamer J,Leitner W,Hammerl P,et al.Designing immune responses with genetic immunization and immunostimulatory DNA sequences.Endocr Regul,2001, 35:143~166.
26 Hartl A,Kiesslich J,Weiss R,et al.Isoforms of the major allergen of birch pollen induce different immunune responses after genetic immunization.Int Arch Allergy Immunol,1999,120:17~19.
27 Woodruff JF.Involvement of T lymphocytes in the pathogenesis of Coxsachie virus B3 heart disease.J Immumol.1974,113:1726~1731.
Hammond EH.Cardiac immune complexes and
28 mononuclear cell subsets in myocarditis:An endomyo-cardial biopsy study.Circulation.1983,68(sⅢ):27~31.
29 Casslng RS.Quantitative evaluation of inflammation in biopsyspecimens from idiopathically failing or irritable hearts:Experience in 80 p ediatric and adult patients.Am Heart J,1985,110: 713~720.
30 沈茜，汪美志，章同华，等。小鼠柯萨基B3病毒心肌炎淋巴细胞亚群及NK细胞的免疫组化研究。中华微生物学和免疫学杂志，1992,12(5):60~63.
31 Shiver JW,Ulmer DB,Donnelly JJ,et al.Humoral and cellular immunities elicited by DNA vaccine:application to the human immunodeficiency virus and influenza.Adv Drug Deliv Rev,1996;21: 19~31.
32 Martins LP,Lau LL,Asano NS,et al.DNA vaccination against persistant virol infection.J Virol,1995, 69: 2574~ 2580.