本文選題：CTL　切入點：小鼠肝炎病毒　出處：《重慶理工大學(xué)》2010年碩士論文

【摘要】：方法: 1.采用超基序、量化基序,并結(jié)合人工神經(jīng)網(wǎng)絡(luò)預(yù)測方案,對小鼠III型肝炎病毒(MHV-3)刺突蛋白(S蛋白)H-2KK限制性CTL表位進行分析。并借助SYBYL7.3及Insight II分子模擬軟件進行CTL表位肽結(jié)構(gòu)特征與其親和力間的3D-QSAR研究,從結(jié)果中選取得分較高的4條MHV-3表位肽進行下一步實驗驗證。 2.采用固相合成技術(shù)合成CTL候選表位肽、純化、分子量鑒定。 3.運用TAP缺陷H-2KK陽性的T2KK細(xì)胞對合成的候選肽與H-2KK分子進行親和力分析。 4.用上述篩選的候選表位肽用不完全福氏佐劑乳化后,免疫C3H小鼠三次后,取其脾淋巴細(xì)胞作為效應(yīng)細(xì)胞,采用標(biāo)準(zhǔn)的LDH乳酸脫氫酶方法檢測小鼠III型肝炎病毒特異性CTL對H-2KK陽性的靶細(xì)胞的免疫殺傷效應(yīng);采用ELISPOT技術(shù)檢測小鼠III型肝炎病毒特異性效應(yīng)細(xì)胞IFN-γ的分泌能力。 結(jié)果: 1.采用超基序和量化基序,并結(jié)合人工神經(jīng)網(wǎng)絡(luò)方法聯(lián)合預(yù)測出4條得分最高的小鼠III型肝炎病毒表位肽:Mhv141-148(IEPYNGVI),Mhv306-313(YELSGYTV),Mhv228-235(FSVYIGDI),Mhv101-108(NDGIFAKV);分子模擬技術(shù)模擬表位肽與MHC分子結(jié)合的空間構(gòu)象,并分析結(jié)合參數(shù),結(jié)果表明預(yù)測出的四條表位肽均符合多肽表位與MHC分子結(jié)合標(biāo)準(zhǔn)。 2.采用固相合成技術(shù)合成了上述4條CTL候選表位肽,高效液相色譜及質(zhì)譜進行純度鑒定,結(jié)果表明合成的4條候選肽純度均在95%以上。 3. TAP缺陷且H-2KK陽性的T2-KK細(xì)胞進行CTL候選表位肽與MHC分子親和力分析,表明4條候選肽中的MHV-3(141-148)及MHV-3(306-313)能與H-2KK有效結(jié)合。 4.為了研究MHV-3(141-148)及MHV-3(306-313)小鼠III型肝炎病毒表位肽體內(nèi)誘導(dǎo)的免疫效應(yīng),我們先將MHV-3(141-148)及MHV-3(306-313)與福氏不完全佐劑乳化,然后分別免疫C3H/Hej小鼠,每周一次,共免疫三次后,取小鼠脾淋巴細(xì)胞作為效應(yīng)細(xì)胞,以H-2KK陽性的L929細(xì)胞負(fù)載相應(yīng)多肽作為靶細(xì)胞,用LDH方法檢測殺傷效應(yīng),實驗結(jié)果表明,小鼠III型肝炎病毒表位特異性的CTL對小鼠III型肝炎病毒陽性且H-2KK陽性的靶細(xì)胞具有明顯的殺傷效應(yīng)。ELISPOT技術(shù)檢測小鼠III型肝炎病毒抗原表位MHV-3(141-148)及MHV-3(306-313)可以促進效應(yīng)細(xì)胞IFN-γ釋放,提示小鼠III型肝炎病毒表位可以促進非特異性的抗病毒免疫效應(yīng)。 結(jié)論: 1.采用生物信息學(xué)并結(jié)合實驗技術(shù)首次從小鼠III型肝炎病毒S蛋白全長氨基酸序列中篩選出2條受H-2KK限制的CTL表位,MHV-3141-14(8IEPYNGVI)和Mhv306-313(YELSGYTV)。 2.從體外及動物實驗兩方面證實小鼠III型肝炎病毒抗原表位MHV-3141-148和Mhv306-313可以誘導(dǎo)產(chǎn)生小鼠III型肝炎病毒特異性的CTL,對小鼠III型肝炎病毒陽性且H-2KK相匹配的靶細(xì)胞具有很強的免疫殺傷活性,提示小鼠III型肝炎病毒抗原表位誘導(dǎo)的CTL反應(yīng)是小鼠III型肝炎病毒特異且H-2KK限制。 3.利用動物實驗證實小鼠III型肝炎病毒抗原表位MHV-3141-148(IEPYNGVI)和Mhv306-313(YELSGYTV)可以促進效應(yīng)細(xì)胞IFN-γ釋放。 以上研究表明,小鼠III型肝炎病毒特異性表位MHV-3141-148(IEPYNGVI)和Mhv306-313(YELSGYTV)不僅能激發(fā)機體特異性的抗病毒效應(yīng),而且還能誘導(dǎo)一個非特異性抗病毒效應(yīng),這種小鼠III型肝炎病毒多肽疫苗具有廣譜,高效、特異、安全的優(yōu)點。本研究從動物在體實驗初步證實了肝炎病毒多條表位多肽疫苗臨床應(yīng)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.

【學(xué)位授予單位】：重慶理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392

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相關(guān)期刊論文 前1條

1 朱波,陳正堂,程曉明,林治華,吳玉章;腫瘤抗原TRAG-3 HLA-A2.1限制性CTL表位的鑒定[J];免疫學(xué)雜志;2004年06期

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本文編號：1697397