人乳头瘤病毒16型E5与IL-12联合基因疫苗的免疫效果研究

发布时间：2018-04-12 07:18

  本文选题：人乳头瘤病毒16型(HPV16) + E5　； 参考：《南华大学》2011年硕士论文

【摘要】：目的: 构建人乳头瘤病毒16型(HPV16)E5蛋白真核与原核表达载体,分析E5与IL-12联合基因疫苗接种BALB/c小鼠后的免疫活性,为HPV防治性疫苗的研制奠定前期试验基础。 方法: (1)运用PRIMER5.0软件设计HPV16 E5基因特异性引物,PCR扩增E5基因,经BamHI、EcoRI或NotI双酶切后将其分别连接入pcDNA3.1(+)或pGEX-6P-1相应酶切位点;PCR及酶切鉴定插入片段,筛选阳性克隆进行DNA序列分析,构建真核表达载体pcDNA3.1(+)/E5与原核表达载体pGEX-6P-1/E5。 (2)将质粒pGEX-6P-1/E5转化E.coli BL21,用IPTG进行诱导表达,纯化表达产物,Western- blot检测GST-HPV16 E5纯化产物。将质粒pcDNA3.1(+)/E5转染HeLa细胞,利用RT-PCR测定HPV16 E5 mRNA表达。 (3)将50只BALB/c小鼠随机分为pcDNA3.1(+)/E5组、pcDNA3.1(+)/E5+ pcDNA 3.1(+)/IL-12组、pcDNA3.1(+)/IL-12组、pcDNA3.1(+)组和PBS空白组,每组10只。将100μg质粒或100μL PBS分别通过肌肉接种小鼠,隔2周接种1次,共接种4次。每次免疫前一天收集血清。末次免疫后第14天收集小鼠血清后,取小鼠脾脏制备脾细胞悬液并培养;另取小鼠股四头肌制成石蜡切片。 (4) ELISA间接法测定小鼠血清中抗HPV16 E5特异性IgG水平,ELISA双抗体夹心法检测小鼠脾淋巴细胞培养上清中IFN-γ和IL-4含量,MTT比色法检测脾淋巴细胞增殖反应,免疫组织化学法检测E5蛋白在小鼠肌肉组织中的表达情况。 结果: (1) DNA测序结果显示PCR扩增得到252bp HPV16 E5基因片段,并分别连接入pcDNA3.1(+)与pGEX-6P-1载体。构建的pcDNA3.1(+)/E5真核表达载体在HeLa细胞表达了HPV16 E5 mRNA。 (2)构建的pGEX-6P-1/E5原核表达载体在E.coli BL21中表达分子量约35kDa GST-HPV16 E5融合蛋白;Western-blot检测该重组蛋白能与抗HPV16 E5抗体结合,表达的融合蛋白分子量约35kDa。 (3)联合基因疫苗组[pcDNA3.1(+)/E5+pcDNA3.1(+)/IL-12]和单基因疫苗组[pcDNA3.1(+)/E5]血清IgG抗体水平随免疫时间的延长呈上升趋势;最后一次免疫的血清IgG A450值分别为(0.771±0.051)和(0.330±0.078),明显高于pcDNA3.1(+)组(0.080±0.035)、pcDNA3.1(+)/IL-12组(0.110±0.015)和PBS组(0.078±0.020) (P0.01);且联合基因疫苗组显著高于单基因疫苗组(P0.01)。 (4)联合基因疫苗组和单基因疫苗组脾细胞培养上清中IFN-γ和IL-4含量最高,分别为(352.89±36.76 pg/ml、680.23±36.04 pg/ml)和(206.53±15.40 pg/ml、359.94±48.23pg/ml) ,均明显高于pcDNA3.1(+)组(18.04±2.24 pg /ml、40.41±4.11pg/ml)、pcDNA3.1(+)/IL-12组(42.15±4.89pg/ml、177.59±24.33pg/ml)、pcDNA3.1(+)组(18.04±2.24pg/ml、40.41±4.11pg/ml)、PBS组(14.98±2.03 pg/ml、25.73±2.02 pg/ml) (P0.01 );且联合基因疫苗组显著高于单基因疫苗组(P0.01)。联合基因疫苗组和单基因疫苗组脾淋巴细胞刺激指数(SI)分别为(2.14±0.27)和(1.85±0.11),显著高于pcDNA3.1(+)组(1.19±0.07)、pcDNA3.1(+)/IL-12组(1.24±0.12)和PBS组(1.13±0.15) (P0.01);联合基因疫苗组与单基因疫苗组比较,SI差异无统计学意义(P0.05)。小鼠股四头肌组织中有HPV16 E5蛋白的表达。 结论: (1)构建的真核表达载体pcDNA3.1(+)/E5能在真核细胞中表达HPV16 E5。 (2)构建的原核表达载体pGEX-6P-1/E5能在E.coli表达融合蛋白GST-HPV16 E5,并具有良好的抗原性。 (3) HPV16 E5单基因疫苗以及与IL-12联合基因疫苗均能刺激机体产生较强的细胞免疫和体液免疫应答,且联合基因疫苗优于单基因疫苗。
[Abstract]:Objective:
Objective to construct eukaryotic and prokaryotic expression vector of human papillomavirus type 16 (HPV16) E5 protein, analyze the immune activity after inoculation of E5 and IL-12 combined vaccine in BALB/c mice, and lay a preliminary experimental basis for the development of HPV vaccine.
Method:
(1) using PRIMER5.0 software to design specific primers of HPV16 E5 gene, PCR E5 gene was amplified by BamHI, EcoRI or NotI after double digestion which were inserted into pcDNA3.1 (+) or pGEX-6P-1 corresponding enzyme sites; PCR enzyme digestion and inserted fragments. The positive clones were screened by DNA sequence analysis, was constructed the eukaryotic expression vector pcDNA3.1 (+) /E5 and prokaryotic expression vector pGEX-6P-1/E5.
(2) plasmid pGEX-6P-1/E5 was transformed into E.coli BL21, induced by IPTG, purified and expressed. Western- blot was used to detect GST-HPV16 E5 purification products. Plasmid pcDNA3.1 (+) /E5 was transfected into the HeLa cells, and the expression was detected by pcDNA3.1.
(3) 50 BALB/c mice were randomly divided into pcDNA3.1 (+ /E5) group, pcDNA3.1 (+) /E5+ pcDNA 3.1 (+) /IL-12 group, /IL-12 group, pcDNA3.1 (+) pcDNA3.1 (+) group and PBS control group, 10 rats in each group. 100 g or 100 L plasmid respectively by PBS the muscle of mice. After 2 weeks of inoculation were 1 times, 4 times a day before inoculation. Each immune sera were collected. Serum were collected fourteenth days after the last immunization, the preparation of spleen cell suspension of mouse spleen and culture; the other mice stock four muscles made into paraffin sections.
(4) determination of anti HPV16 E5 in mice serum specific IgG level ELISA indirect method, detection of mouse spleen lymphocytes ELISA double antibody sandwich cultured IFN- gamma and IL-4 content in the supernatant of spleen lymphocyte proliferation by MTT assay, the expression of E5 protein was detected by immunohistochemistry in muscle of mice.
Result:
(1) DNA sequencing results showed that 252bp HPV16 E5 gene fragment was amplified by PCR and connected to pcDNA3.1 (+) and pGEX-6P-1 vector respectively. The pcDNA3.1 (+) /E5 eukaryotic expression vector was constructed in HeLa cells, and expressed the HPV16 /E5.
(2) the pGEX-6P-1/E5 prokaryotic expression vector was constructed in E.coli BL21 and expressed in 35kDa BL21 E5 fusion protein. Western-blot detected that the recombinant protein could bind to anti HPV16 E5 antibody and the molecular weight of fusion protein was about 35kDa..
(3) gene vaccine group [pcDNA3.1 (+) /E5+pcDNA3.1 (+ /IL-12]) and Dan Jiyin [pcDNA3.1 (+) /E5] vaccine group IgG antibody levels in serum with immune time increased; the last time the immune serum IgG A450 = (0.771 + 0.051) and (0.330 + 0.078) was significantly higher than that of pcDNA3.1. (+) group (0.080 + 0.035), pcDNA3.1 (+) /IL-12 group (0.110 + 0.015) and PBS group (0.078 + 0.020) (P0.01); and the combination of gene vaccine group was significantly higher than that of single gene vaccine group (P0.01).
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