小鼠III型肝炎病毒H-2K~K限制性CTL表位预测及初步鉴定

发布时间：2018-04-01 21:37

本文选题：CTL　切入点：小鼠肝炎病毒　出处：《重庆理工大学》2010年硕士论文

【摘要】：方法: 1.采用超基序、量化基序,并结合人工神经网络预测方案,对小鼠III型肝炎病毒(MHV-3)刺突蛋白(S蛋白)H-2KK限制性CTL表位进行分析。并借助SYBYL7.3及Insight II分子模拟软件进行CTL表位肽结构特征与其亲和力间的3D-QSAR研究,从结果中选取得分较高的4条MHV-3表位肽进行下一步实验验证。 2.采用固相合成技术合成CTL候选表位肽、纯化、分子量鉴定。 3.运用TAP缺陷H-2KK阳性的T2KK细胞对合成的候选肽与H-2KK分子进行亲和力分析。 4.用上述筛选的候选表位肽用不完全福氏佐剂乳化后,免疫C3H小鼠三次后,取其脾淋巴细胞作为效应细胞,采用标准的LDH乳酸脱氢酶方法检测小鼠III型肝炎病毒特异性CTL对H-2KK阳性的靶细胞的免疫杀伤效应;采用ELISPOT技术检测小鼠III型肝炎病毒特异性效应细胞IFN-γ的分泌能力。结果: 1.采用超基序和量化基序,并结合人工神经网络方法联合预测出4条得分最高的小鼠III型肝炎病毒表位肽:Mhv141-148(IEPYNGVI),Mhv306-313(YELSGYTV),Mhv228-235(FSVYIGDI),Mhv101-108(NDGIFAKV);分子模拟技术模拟表位肽与MHC分子结合的空间构象,并分析结合参数,结果表明预测出的四条表位肽均符合多肽表位与MHC分子结合标准。 2.采用固相合成技术合成了上述4条CTL候选表位肽,高效液相色谱及质谱进行纯度鉴定,结果表明合成的4条候选肽纯度均在95%以上。 3. TAP缺陷且H-2KK阳性的T2-KK细胞进行CTL候选表位肽与MHC分子亲和力分析,表明4条候选肽中的MHV-3(141-148)及MHV-3(306-313)能与H-2KK有效结合。 4.为了研究MHV-3(141-148)及MHV-3(306-313)小鼠III型肝炎病毒表位肽体内诱导的免疫效应,我们先将MHV-3(141-148)及MHV-3(306-313)与福氏不完全佐剂乳化,然后分别免疫C3H/Hej小鼠,每周一次,共免疫三次后,取小鼠脾淋巴细胞作为效应细胞,以H-2KK阳性的L929细胞负载相应多肽作为靶细胞,用LDH方法检测杀伤效应,实验结果表明,小鼠III型肝炎病毒表位特异性的CTL对小鼠III型肝炎病毒阳性且H-2KK阳性的靶细胞具有明显的杀伤效应。ELISPOT技术检测小鼠III型肝炎病毒抗原表位MHV-3(141-148)及MHV-3(306-313)可以促进效应细胞IFN-γ释放,提示小鼠III型肝炎病毒表位可以促进非特异性的抗病毒免疫效应。结论: 1.采用生物信息学并结合实验技术首次从小鼠III型肝炎病毒S蛋白全长氨基酸序列中筛选出2条受H-2KK限制的CTL表位,MHV-3141-14(8IEPYNGVI)和Mhv306-313(YELSGYTV)。 2.从体外及动物实验两方面证实小鼠III型肝炎病毒抗原表位MHV-3141-148和Mhv306-313可以诱导产生小鼠III型肝炎病毒特异性的CTL,对小鼠III型肝炎病毒阳性且H-2KK相匹配的靶细胞具有很强的免疫杀伤活性,提示小鼠III型肝炎病毒抗原表位诱导的CTL反应是小鼠III型肝炎病毒特异且H-2KK限制。 3.利用动物实验证实小鼠III型肝炎病毒抗原表位MHV-3141-148(IEPYNGVI)和Mhv306-313(YELSGYTV)可以促进效应细胞IFN-γ释放。以上研究表明,小鼠III型肝炎病毒特异性表位MHV-3141-148(IEPYNGVI)和Mhv306-313(YELSGYTV)不仅能激发机体特异性的抗病毒效应,而且还能诱导一个非特异性抗病毒效应,这种小鼠III型肝炎病毒多肽疫苗具有广谱,高效、特异、安全的优点。本研究从动物在体实验初步证实了肝炎病毒多条表位多肽疫苗临床应用的可能性。
[Abstract]:Method:
1. by supermotif, quantitative motif, and combined with artificial neural network forecasting method of mouse hepatitis III virus (MHV-3) spike protein (S protein) H-2KK restricted CTL epitopes were analyzed. With the help of SYBYL7.3 and Insight II molecular simulation software 3D-QSAR to study the peptide structure with the affinity between CTL table, select higher scores from the results of the 4 MHV-3 epitope peptide for the next experiment.
2. the solid phase synthesis technique was used to synthesize the CTL candidate epitope peptide, purification and molecular weight identification.
3. the affinity analysis of the synthesized candidate peptides and H-2KK molecules was carried out by using TAP deficient H-2KK positive T2KK cells.
4. with the screening of candidate epitope peptides with incomplete Freund's adjuvant, C3H mice were immunized three times, from the spleen lymphocytes were used as effector cells, the immune killing effect of LDH lactic acid dehydrogenase method using standard detection of mouse hepatitis III virus specific CTL on target cells H-2KK positive; secretion by ELISPOT were used for the detection of hepatitis III virus specific effector cells IFN- gamma.
Result:
1. the supermotif and quantitative motif, and combined with artificial neural network method combined with prediction of 4 the highest score of mouse hepatitis III virus epitope peptide Mhv141-148 (IEPYNGVI), Mhv306-313 (YELSGYTV), Mhv228-235 (FSVYIGDI), Mhv101-108 (NDGIFAKV); molecular simulation technology to simulate the conformational epitope peptide and binding of MHC, and combining the analysis of parameters, the results showed that four epitopes predicted are consistent with the peptide epitope with MHC binding standards.
2., the above 4 CTL candidate epitope peptides were synthesized by solid phase synthesis, and identified by HPLC and MS. The results showed that the purity of 4 candidate peptides was above 95%.
3. TAP deficient and H-2KK positive T2-KK cells carry out affinity analysis of CTL candidate epitope peptides and MHC molecules, indicating that MHV-3 (141-148) and MHV-3 (306-313) of 4 candidate peptides can effectively combine with H-2KK.
4. in order to study the MHV-3 (141-148) and MHV-3 (306-313) mice hepatitis III virus epitope peptide induced immune response, we first MHV-3 (141-148) and MHV-3 (306-313) and Freund's incomplete adjuvant emulsion, and then C3H/Hej mice were immunized once a week, a total of three times after immunization of mice. Spleen lymphocytes were used as effector cells in H-2KK positive L929 cells loaded with corresponding peptides as target cells, using LDH method to detect the killing effect, the experimental results show that the mouse hepatitis III virus epitope specific CTL has obvious killing effect of.ELISPOT were used for the detection of hepatitis III virus MHV-3 antigen epitope of hepatitis III virus in mice positive and H-2KK positive cells (141-148) and MHV-3 (306-313) IFN- cells can promote the effect of gamma release, suggesting that the mouse hepatitis III virus epitope can promote non-specific antiviral immune effect.
Conclusion:
1., using bioinformatics and experimental techniques, we first screened out 2 H-2KK restricted CTL epitopes, MHV-3141-14 (8IEPYNGVI) and Mhv306-313 (YELSGYTV), from the full-length amino acid sequence of mouse hepatitis III virus S protein.
2. from the two in vitro and animal experiments confirmed that mice hepatitis III virus antigen epitopes of MHV-3141-148 and Mhv306-313 can induce mouse hepatitis III virus specific CTL target cells matched on mouse hepatitis III virus positive and H-2KK has strong killing activity of immune mice, suggesting that hepatitis III virus antigen a reaction induced CTL mouse hepatitis III virus specific and H-2KK limit.
3. using animal experiments, it is proved that the antigen epitopes of hepatitis III virus (IEPYNGVI) and Mhv306-313 (YELSGYTV) in mice can promote the release of IFN- gamma in the effector cells.
These studies show that mouse hepatitis III virus specific epitope MHV-3141-148 (IEPYNGVI) and Mhv306-313 (YELSGYTV) antiviral effect can not only stimulate the body specificity, but also induced a nonspecific antiviral effect, this mouse hepatitis III virus peptide vaccine has broad spectrum, efficient, specific, safe advantages. From this study the animal in vivo experiments confirmed that hepatitis B virus multi epitope peptide vaccine and the possibility of clinical application.

【学位授予单位】：重庆理工大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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