發(fā)布時(shí)間：2018-04-12 07:18

  本文選題：人乳頭瘤病毒16型(HPV16) + E5��； 參考：《南華大學(xué)》2011年碩士論文

【摘要】：目的: 構(gòu)建人乳頭瘤病毒16型(HPV16)E5蛋白真核與原核表達(dá)載體,分析E5與IL-12聯(lián)合基因疫苗接種BALB/c小鼠后的免疫活性,為HPV防治性疫苗的研制奠定前期試驗(yàn)基礎(chǔ)。 方法: (1)運(yùn)用PRIMER5.0軟件設(shè)計(jì)HPV16 E5基因特異性引物,PCR擴(kuò)增E5基因,經(jīng)BamHI、EcoRI或NotI雙酶切后將其分別連接入pcDNA3.1(+)或pGEX-6P-1相應(yīng)酶切位點(diǎn);PCR及酶切鑒定插入片段,篩選陽性克隆進(jìn)行DNA序列分析,構(gòu)建真核表達(dá)載體pcDNA3.1(+)/E5與原核表達(dá)載體pGEX-6P-1/E5。 (2)將質(zhì)粒pGEX-6P-1/E5轉(zhuǎn)化E.coli BL21,用IPTG進(jìn)行誘導(dǎo)表達(dá),純化表達(dá)產(chǎn)物,Western- blot檢測(cè)GST-HPV16 E5純化產(chǎn)物。將質(zhì)粒pcDNA3.1(+)/E5轉(zhuǎn)染HeLa細(xì)胞,利用RT-PCR測(cè)定HPV16 E5 mRNA表達(dá)。 (3)將50只BALB/c小鼠隨機(jī)分為pcDNA3.1(+)/E5組、pcDNA3.1(+)/E5+ pcDNA 3.1(+)/IL-12組、pcDNA3.1(+)/IL-12組、pcDNA3.1(+)組和PBS空白組,每組10只。將100μg質(zhì)�；�100μL PBS分別通過肌肉接種小鼠,隔2周接種1次,共接種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,取小鼠脾臟制備脾細(xì)胞懸液并培養(yǎng);另取小鼠股四頭肌制成石蠟切片。 (4) ELISA間接法測(cè)定小鼠血清中抗HPV16 E5特異性IgG水平,ELISA雙抗體夾心法檢測(cè)小鼠脾淋巴細(xì)胞培養(yǎng)上清中IFN-γ和IL-4含量,MTT比色法檢測(cè)脾淋巴細(xì)胞增殖反應(yīng),免疫組織化學(xué)法檢測(cè)E5蛋白在小鼠肌肉組織中的表達(dá)情況。 結(jié)果: (1) DNA測(cè)序結(jié)果顯示PCR擴(kuò)增得到252bp HPV16 E5基因片段,并分別連接入pcDNA3.1(+)與pGEX-6P-1載體。構(gòu)建的pcDNA3.1(+)/E5真核表達(dá)載體在HeLa細(xì)胞表達(dá)了HPV16 E5 mRNA。 (2)構(gòu)建的pGEX-6P-1/E5原核表達(dá)載體在E.coli BL21中表達(dá)分子量約35kDa GST-HPV16 E5融合蛋白;Western-blot檢測(cè)該重組蛋白能與抗HPV16 E5抗體結(jié)合,表達(dá)的融合蛋白分子量約35kDa。 (3)聯(lián)合基因疫苗組[pcDNA3.1(+)/E5+pcDNA3.1(+)/IL-12]和單基因疫苗組[pcDNA3.1(+)/E5]血清IgG抗體水平隨免疫時(shí)間的延長呈上升趨勢(shì);最后一次免疫的血清IgG A450值分別為(0.771±0.051)和(0.330±0.078),明顯高于pcDNA3.1(+)組(0.080±0.035)、pcDNA3.1(+)/IL-12組(0.110±0.015)和PBS組(0.078±0.020) (P0.01);且聯(lián)合基因疫苗組顯著高于單基因疫苗組(P0.01)。 (4)聯(lián)合基因疫苗組和單基因疫苗組脾細(xì)胞培養(yǎng)上清中IFN-γ和IL-4含量最高,分別為(352.89±36.76 pg/ml、680.23±36.04 pg/ml)和(206.53±15.40 pg/ml、359.94±48.23pg/ml) ,均明顯高于pcDNA3.1(+)組(18.04±2.24 pg /ml、40.41±4.11pg/ml)、pcDNA3.1(+)/IL-12組(42.15±4.89pg/ml、177.59±24.33pg/ml)、pcDNA3.1(+)組(18.04±2.24pg/ml、40.41±4.11pg/ml)、PBS組(14.98±2.03 pg/ml、25.73±2.02 pg/ml) (P0.01 );且聯(lián)合基因疫苗組顯著高于單基因疫苗組(P0.01)。聯(lián)合基因疫苗組和單基因疫苗組脾淋巴細(xì)胞刺激指數(shù)(SI)分別為(2.14±0.27)和(1.85±0.11),顯著高于pcDNA3.1(+)組(1.19±0.07)、pcDNA3.1(+)/IL-12組(1.24±0.12)和PBS組(1.13±0.15) (P0.01);聯(lián)合基因疫苗組與單基因疫苗組比較,SI差異無統(tǒng)計(jì)學(xué)意義(P0.05)。小鼠股四頭肌組織中有HPV16 E5蛋白的表達(dá)。 結(jié)論: (1)構(gòu)建的真核表達(dá)載體pcDNA3.1(+)/E5能在真核細(xì)胞中表達(dá)HPV16 E5。 (2)構(gòu)建的原核表達(dá)載體pGEX-6P-1/E5能在E.coli表達(dá)融合蛋白GST-HPV16 E5,并具有良好的抗原性。 (3) HPV16 E5單基因疫苗以及與IL-12聯(lián)合基因疫苗均能刺激機(jī)體產(chǎn)生較強(qiáng)的細(xì)胞免疫和體液免疫應(yīng)答,且聯(lián)合基因疫苗優(yōu)于單基因疫苗。
[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).
(4)鑱斿悎鍩哄洜鐤嫍緇勫拰鍗曞熀鍥犵柅鑻楃粍鑴劇粏鑳?yōu)鍩瑰呏M笂娓呬腑IFN-緯鍜孖L-4鍚噺鏈，

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