结核亚单位疫苗LT70-DPC安全性初步评价与融合蛋白MAR和MRA的构建

发布时间：2018-05-31 17:59

本文选题：结核 + 亚单位疫苗　；参考：《兰州大学》2017年硕士论文

【摘要】：第一章结核亚单位疫苗LT70-DPC安全性初步评价目的:对以阳离子脂质体二甲基三十六烷基铵(dimo-thylidioctyl ammonium bromide,DDA)、聚肌胞苷(polyriboinosinic-polyribocytidylic acid,Poly I:C)与胆固醇(cholesterol)复合佐剂(简称为DPC)为佐剂的结核融合蛋白ESAT6-Ag85B-MPT64(190-198)-MTB8.4-Rv2626c(LT70)亚单位疫苗的毒理学进行研究,以初步评价其安全性。方法:1.急性毒性试验,分别将LT70-DPC亚单位疫苗与佐剂DDA的低剂量与高剂量(分别相当于人用剂量的10 000倍和40 000倍)经皮下、腹腔两种途径1次给予小鼠,观察毒性反应和死亡情况等,同时推算小鼠对LT70-DPC亚单位疫苗的最大耐受剂量(maximum tolerated dose,MTD)。2.异常毒性试验,小鼠和豚鼠腹腔1次注射疫苗,观察有无异常反应、死亡情况及体重变化。3.全身过敏试验,豚鼠致敏后静脉快速注射攻击,观察有无过敏反应。结果:1.急性毒性试验,将两种浓度的疫苗经皮下与腹腔两种途径注射小鼠后,均未引起小鼠的异常反应和死亡,小鼠体重增加。小鼠对LT70-DPC亚单位疫苗的最大耐剂受量(MTD)为:LT70蛋白6 600μg/kg,DDA166 600μg/kg,Poly I:C33 200μg/kg,胆固醇50 600μg/kg。2.异常毒性试验,腹腔注射7d后,小鼠与豚鼠全部健存,体重增加,且均未出现异常症状。3.全身过敏试验,疫苗组均未出现过敏反应。结论:急性毒性试验,异常毒性试验与全身过敏试验的结果显示两种浓度的疫苗均未检测到毒副作用,表明LT70-DPC蛋白亚单位疫苗安全性好。第二章结核亚单位疫苗融合蛋白的构建目的:为使实验室前期已构建的无标签的融合蛋白ESAT6-Ag85BMPT64(190-198)-MTB8.4-Rv2626c(LT70)更易纯化,对其进行优化改造。同时为研究不同融合蛋白的表达与纯化特性,本研究选用结核分枝杆菌早期分泌蛋白Ag85B和ESAT6家族成员MTB10.4,以及潜伏期抗原Rv1738,克隆构建无标签结核分枝杆菌融合蛋白MTB10.4-Ag85B-Rv1738(MAR)与MTB10.4-Rv1738-Ag85B(MRA)。方法:通过结构预测,对LT70蛋白中的Ag85B基因进行定点突变,将第83个氨基酸I-ATA异亮氨酸突变为为CGT-R精氨酸,第140个氨基酸亮氨酸L-TTG突变为AAA-K赖氨酸,以减弱蛋白的疏水性。应用分子克隆技术设计含有不同酶切位点的引物,以H37Rv-DNA为模板,PCR扩增相应基因片段,将抗原基因MTB10.4、Ag85B和Rv1738依次插入到表达载体p ET-30a(+)的多克隆位点中,构建重组质粒p ET30a-MTB10.4-Ag85B-Rv1738与p ET30aMTB10.4-Rv1738-Ag85B;将重组质粒转入大肠杆菌BL-21(DE3),IPTG诱导表达,采用疏水作用色谱层析(Hydrophobic interaction chromatography,HIC)和离子交换色谱层析((Ion exchange chromat-ography,IEX)等技术进行融合蛋白的纯化。结果:突变后的LT70蛋白相较之前,疏水性仍然较强,结合到疏水作用层析柱后不易洗脱。构建的融合蛋白MAR和MRA经PCR扩增获得的基因序列与Gen Bank中完全一致,在大肠杆菌中表达产物分子量与预计相同,约53KD,二者分别以包涵体和上清表达为主,应用离子交换色谱层析(IEX)和疏水作用色谱层析(HIC)等方法初步纯化蛋白。结论:此次对LT70蛋白的优化改造并未很好的改善其疏水性,其改造方法有待进一步研究。同时成功构建了不带标签的结核融合蛋MTB10.4-Ag85BRv1738(MAR)与MTB10.4-Rv1738-Ag85B(MRA),且利用不同的色谱柱使融合蛋白得到了初步的纯化,为后续免疫原性检测及动物保护试验做好准备。
[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.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392

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