發(fā)布時(shí)間：2018-05-31 17:59

  本文選題：結(jié)核 + 亞單位疫苗��； 參考：《蘭州大學(xué)》2017年碩士論文

【摘要】：第一章結(jié)核亞單位疫苗LT70-DPC安全性初步評(píng)價(jià)目的:對(duì)以陽(yáng)離子脂質(zhì)體二甲基三十六烷基銨(dimo-thylidioctyl ammonium bromide,DDA)、聚肌胞苷(polyriboinosinic-polyribocytidylic acid,Poly I:C)與膽固醇(cholesterol)復(fù)合佐劑(簡(jiǎn)稱為DPC)為佐劑的結(jié)核融合蛋白ESAT6-Ag85B-MPT64(190-198)-MTB8.4-Rv2626c(LT70)亞單位疫苗的毒理學(xué)進(jìn)行研究,以初步評(píng)價(jià)其安全性。方法:1.急性毒性試驗(yàn),分別將LT70-DPC亞單位疫苗與佐劑DDA的低劑量與高劑量(分別相當(dāng)于人用劑量的10 000倍和40 000倍)經(jīng)皮下、腹腔兩種途徑1次給予小鼠,觀察毒性反應(yīng)和死亡情況等,同時(shí)推算小鼠對(duì)LT70-DPC亞單位疫苗的最大耐受劑量(maximum tolerated dose,MTD)。2.異常毒性試驗(yàn),小鼠和豚鼠腹腔1次注射疫苗,觀察有無(wú)異常反應(yīng)、死亡情況及體重變化。3.全身過(guò)敏試驗(yàn),豚鼠致敏后靜脈快速注射攻擊,觀察有無(wú)過(guò)敏反應(yīng)。結(jié)果:1.急性毒性試驗(yàn),將兩種濃度的疫苗經(jīng)皮下與腹腔兩種途徑注射小鼠后,均未引起小鼠的異常反應(yīng)和死亡,小鼠體重增加。小鼠對(duì)LT70-DPC亞單位疫苗的最大耐劑受量(MTD)為:LT70蛋白6 600μg/kg,DDA166 600μg/kg,Poly I:C33 200μg/kg,膽固醇50 600μg/kg。2.異常毒性試驗(yàn),腹腔注射7d后,小鼠與豚鼠全部健存,體重增加,且均未出現(xiàn)異常癥狀。3.全身過(guò)敏試驗(yàn),疫苗組均未出現(xiàn)過(guò)敏反應(yīng)。結(jié)論:急性毒性試驗(yàn),異常毒性試驗(yàn)與全身過(guò)敏試驗(yàn)的結(jié)果顯示兩種濃度的疫苗均未檢測(cè)到毒副作用,表明LT70-DPC蛋白亞單位疫苗安全性好。第二章結(jié)核亞單位疫苗融合蛋白的構(gòu)建目的:為使實(shí)驗(yàn)室前期已構(gòu)建的無(wú)標(biāo)簽的融合蛋白ESAT6-Ag85BMPT64(190-198)-MTB8.4-Rv2626c(LT70)更易純化,對(duì)其進(jìn)行優(yōu)化改造。同時(shí)為研究不同融合蛋白的表達(dá)與純化特性,本研究選用結(jié)核分枝桿菌早期分泌蛋白Ag85B和ESAT6家族成員MTB10.4,以及潛伏期抗原Rv1738,克隆構(gòu)建無(wú)標(biāo)簽結(jié)核分枝桿菌融合蛋白MTB10.4-Ag85B-Rv1738(MAR)與MTB10.4-Rv1738-Ag85B(MRA)。方法:通過(guò)結(jié)構(gòu)預(yù)測(cè),對(duì)LT70蛋白中的Ag85B基因進(jìn)行定點(diǎn)突變,將第83個(gè)氨基酸I-ATA異亮氨酸突變?yōu)闉镃GT-R精氨酸,第140個(gè)氨基酸亮氨酸L-TTG突變?yōu)锳AA-K賴氨酸,以減弱蛋白的疏水性。應(yīng)用分子克隆技術(shù)設(shè)計(jì)含有不同酶切位點(diǎn)的引物,以H37Rv-DNA為模板,PCR擴(kuò)增相應(yīng)基因片段,將抗原基因MTB10.4、Ag85B和Rv1738依次插入到表達(dá)載體p ET-30a(+)的多克隆位點(diǎn)中,構(gòu)建重組質(zhì)粒p ET30a-MTB10.4-Ag85B-Rv1738與p ET30aMTB10.4-Rv1738-Ag85B;將重組質(zhì)粒轉(zhuǎn)入大腸桿菌BL-21(DE3),IPTG誘導(dǎo)表達(dá),采用疏水作用色譜層析(Hydrophobic interaction chromatography,HIC)和離子交換色譜層析((Ion exchange chromat-ography,IEX)等技術(shù)進(jìn)行融合蛋白的純化。結(jié)果:突變后的LT70蛋白相較之前,疏水性仍然較強(qiáng),結(jié)合到疏水作用層析柱后不易洗脫。構(gòu)建的融合蛋白MAR和MRA經(jīng)PCR擴(kuò)增獲得的基因序列與Gen Bank中完全一致,在大腸桿菌中表達(dá)產(chǎn)物分子量與預(yù)計(jì)相同,約53KD,二者分別以包涵體和上清表達(dá)為主,應(yīng)用離子交換色譜層析(IEX)和疏水作用色譜層析(HIC)等方法初步純化蛋白。結(jié)論:此次對(duì)LT70蛋白的優(yōu)化改造并未很好的改善其疏水性,其改造方法有待進(jìn)一步研究。同時(shí)成功構(gòu)建了不帶標(biāo)簽的結(jié)核融合蛋MTB10.4-Ag85BRv1738(MAR)與MTB10.4-Rv1738-Ag85B(MRA),且利用不同的色譜柱使融合蛋白得到了初步的純化,為后續(xù)免疫原性檢測(cè)及動(dòng)物保護(hù)試驗(yàn)做好準(zhǔn)備。
[Abstract]:Chapter 1 the primary evaluation of the LT70-DPC safety of the tuberculous subunit vaccine: the fusion egg with a cationic liposome two methyl thirty-six alkyl ammonium (dimo-thylidioctyl ammonium bromide, DDA), polycytidine (polyriboinosinic-polyribocytidylic acid, Poly I:C) and cholesterol (cholesterol) compound adjuvant (referred to as DPC) as adjuvant The toxicology of the white ESAT6-Ag85B-MPT64 (190-198) -MTB8.4-Rv2626c (LT70) subunit vaccine was studied to evaluate its safety preliminarily. Methods: 1. acute toxicity tests were carried out by the subcutaneous and 1 subcutaneous two pathways of the low and high doses of LT70-DPC subunit vaccine and adjuvant DDA (equivalent to 10000 and 40000 times the amount of human dosage, respectively). The mice were given the toxicity and death, and the maximum tolerance dose of the LT70-DPC subunit vaccine (maximum tolerated dose, MTD).2. abnormal toxicity test was calculated, and the mice and guinea pig abdominal cavity were vaccinated for 1 times. The abnormal reaction, the death condition and the weight change.3. allergy test, the guinea pig sensitized vein were observed. Results: 1. acute toxicity tests, after injection of two kinds of vaccine in two ways of subcutaneous and abdominal cavity, did not cause abnormal reaction and death of mice and the weight of mice increased. The maximum dose tolerance of mice to LT70-DPC subunit vaccine (MTD) was LT70 protein 6600 g/kg, DDA166 600 G/kg, Poly I:C33 200 mu g/kg, cholesterol 50600 g/kg.2. abnormal toxicity test, after intraperitoneal injection of 7D, mice and guinea pigs all survived, weight increased, and no abnormal symptoms of.3. general allergy test, no allergic reaction in the vaccine group. Conclusion: acute toxicity test, abnormal toxicity test and systemic allergy test results showed two kinds of results. The concentration vaccine did not detect the toxic side effects, which showed that the LT70-DPC protein subunit vaccine was safe. Second the purpose of the construction of the fusion protein of the tuberculosis subunit vaccine was to make the unlabelled fusion protein ESAT6-Ag85BMPT64 (190-198) -MTB8.4-Rv2626c (LT70) more easily purified and optimized. In this study, the expression and purification characteristics of different fusion proteins were studied. In this study, the early secretory protein Ag85B of Mycobacterium tuberculosis and ESAT6 family members MTB10.4, as well as latent period antigen Rv1738, were cloned to construct the unlabelled Mycobacterium tuberculosis fusion protein MTB10.4-Ag85B-Rv1738 (MAR) and MTB10.4-Rv1738-Ag85B (MRA). The Ag85B gene in the protein was mutagenesis, mutated eighty-third amino acid I-ATA isoleucine into CGT-R arginine, and 140th amino acid leucine L-TTG mutated to AAA-K lysine to reduce the hydrophobicity of the protein. Molecular cloning technology was used to design primers containing different enzyme cut sites, H37Rv-DNA as a template and PCR amplification of the corresponding gene. Fragment, the antigen genes MTB10.4, Ag85B and Rv1738 were inserted into the polyclonal loci of the expression vector p ET-30a (+), and the recombinant plasmid P ET30a-MTB10.4-Ag85B-Rv1738 and P ET30aMTB10.4-Rv1738-Ag85B were constructed, and the recombinant plasmid was transferred to BL-21 (DE3) of Escherichia coli, and the IPTG induced expression was induced by hydrophobic interaction chromatography chromatography. Romatography, HIC) and the purification of fusion protein by ion exchange chromatography (Ion exchange chromat-ography, IEX). Results: after the mutation of LT70 protein, the hydrophobicity is still stronger, and it is not easy to elute after the hydrophobic interaction chromatography column. The gene sequences obtained by the fusion protein MAR and MRA are amplified by PCR and Gen Ban. In K, the molecular weight of the expression products in Escherichia coli was the same as expected, about 53KD, the two were mainly expressed in inclusion body and supernatant respectively. The protein was purified by the methods of ion exchange chromatography (IEX) and hydrophobic interaction chromatography (HIC). Conclusion: the optimization of the LT70 protein is not better to improve its hydrophobicity. The methods of transformation need to be further studied. At the same time, MTB10.4-Ag85BRv1738 (MAR) and MTB10.4-Rv1738-Ag85B (MRA) were successfully constructed, and the fusion protein was preliminarily purified by different chromatographic columns, which prepared for the subsequent immunogenicity detection and animal protection test.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R392

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