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  本文選題：基因轉(zhuǎn)導 + P16衍生肽�。� 參考：《吉林大學》2014年碩士論文

【摘要】：提高基因治療的效率需要合適的基因載體系統(tǒng)和逐漸發(fā)展的用于增強基因轉(zhuǎn)導化合物的使用。逆轉(zhuǎn)錄病毒載體使外源基因在宿主細胞內(nèi)實現(xiàn)穩(wěn)定、長期的表達而占有很大的優(yōu)勢。 病毒與靶細胞表面的負電荷產(chǎn)生的靜電斥力大大限制了基因轉(zhuǎn)導的效率。用于增強基因轉(zhuǎn)導的化合物從最初的陽離子化合物Poly-L-lysine等發(fā)展至今，商品化的DEAE dextran和polybrene仍被人們廣泛應用于基因轉(zhuǎn)導的實驗中。最近的研究表明，淀粉樣纖維多肽在促進基因轉(zhuǎn)導過程中具有突出的優(yōu)勢，如SEVI和EF-C，已經(jīng)被人們證實可以提高逆轉(zhuǎn)錄病毒基因轉(zhuǎn)導的效率，而且顯著強于DEAE和polybrene。 最近我們發(fā)現(xiàn)合成的HIV-14E10表位多肽P13（Ac-671NWFDITNWLWY-IK683-NH2）及其衍生肽P16（Ac-671NWFDITNWLWYIK683KKK-NH2），P16在P13序列C末端增加三個賴氨酸，也能夠形成淀粉樣纖維結(jié)構(gòu)促進HIV-1感染，并且效率強于SEVI。對P13和P16促感染機理的研究表明，芳香族氨基酸色氨酸和堿性氨基酸賴氨酸對P16的促感染活性很重要。由它們的作用方式來看，淀粉樣纖維和正電性是多肽多聚物行使功能的必要條件。 本文的研究主要是在有利于淀粉樣纖維結(jié)構(gòu)形成的前提下，對P16序列中的酸性氨基酸--天冬氨酸進行突變或增加序列中堿性氨基酸的數(shù)目，以期獲得促感染活性更好的多肽。實驗中合成的多肽有P16-D、P16-K、P16-N、P16-2W和P16-W3K。對合成的多肽進行MS和HPLC的質(zhì)量檢測無誤后，按照P16發(fā)揮最佳感染增強效果的條件，即在無血清條件下感染4h后再補加血清或更換為完全培養(yǎng)基，我們對合成的P16衍生肽的促感染活性進行了研究。綜合考慮促進病毒感染的效率和細胞毒性兩個因素，選用P16-D（Ac-671NW FAITNWLWYIK683K-KK-NH2）為研究對象。 病毒感染實驗結(jié)果表明P16-D與P16增強病毒感染的倍數(shù)分別是13.9和7.5，充分說明改造后的P16-D的促感染活性在P16基礎上提高將近100%，同時與最適作用濃度DEAE和polybrene相比，促感染過程中細胞存活率分別是90%與80%左右，P16-D對靶細胞的毒性更低。 TEM、Tht特異性結(jié)合實驗及EGCG實驗表明P16-D經(jīng)過低速攪拌形成典型的淀粉樣纖維結(jié)構(gòu)，能夠有效地捕獲靶細胞和病毒顆粒，同時也使病毒顆粒通過簡單地低速離心就可以達到濃縮的目的。P16-D淀粉樣纖維的促感染活性對HIV-1以外的包膜病毒—流感病毒及FIV載體系統(tǒng)也適用。 鑒于P16-D淀粉樣多肽也能有效地捕獲流感病毒顆粒；在P16-D存在時，流感病毒的血凝效價比不加多肽組提高4倍；流感病毒感染MDCK細胞后，檢測上清里的病毒量，加肽組明顯高于不加肽組，預示著P16-D淀粉樣多肽將在流感病毒免疫中發(fā)揮特殊的作用。 理想的病毒抗原經(jīng)免疫后應該既能引起體液免疫，又能引起細胞免疫�？紤]到P16-D纖維能夠有效地結(jié)合流感病毒顆粒，在細胞水平上促進流感病毒的感染，我們選擇降低劑量的流感病毒配合P16-D淀粉樣纖維一起使用后，經(jīng)鼻腔接種小鼠后，特異性IgG抗體水平低于正常劑量病毒組，但是分型抗體IgG1的水平比后者要高，IgG2a的水平則與之相當，，表明降低劑量的減毒流感病毒配合P16-D淀粉樣多肽使用，也能夠刺激機體產(chǎn)生有效的免疫應答。 綜上，P16-D優(yōu)越的促感染活性預示著它將在逆轉(zhuǎn)錄病毒感染和基因治療中具有廣闊的應用前景，同時，它在減毒的流感免疫中的應用不僅幫助人們優(yōu)化免疫方案，降低生產(chǎn)成本，而且推動了對淀粉樣纖維多聚物的深入認識。
[Abstract]:In order to improve the efficiency of gene therapy, a suitable gene carrier system and the growing use of gene transducing compounds are needed. Retroviral vectors make foreign genes stable and long-term expression in host cells.
The electrostatic repulsion produced by the negative charges on the surface of the virus and the target cell greatly restricts the efficiency of gene transduction. The commercialized DEAE dextran and Polybrene are still widely used in the experiment of gene transduction from the initial cationic compound Poly-L-lysine, which is used to enhance gene transduction. Recent studies have shown that Amyloid polypeptide has prominent advantages in promoting gene transduction, such as SEVI and EF-C, which have been proved to improve the efficiency of retroviral gene transduction, and are significantly stronger than DEAE and polybrene..
Recently, we found that the synthesized HIV-14E10 epitope polypeptide P13 (Ac-671NWFDITNWLWY-IK683-NH2) and its derivative peptide P16 (Ac-671NWFDITNWLWYIK683KKK-NH2), P16 added three lysine at the C terminal of the P13 sequence, and can also form amyloid fiber structure to promote HIV-1 infection, and the efficiency is stronger than the mechanism of P13 and P16 promoting infection. The aromatic amino acid tryptophan and alkaline amino acid lysine are important for the infection promoting activity of P16. By their way of action, the amyloid fiber and the positive power are the necessary conditions for the function of polypeptide polymers.
The main research of this paper is to mutate the acid amino acid, aspartic acid in the P16 sequence, or increase the number of basic amino acids in the sequence, in order to obtain the polypeptide with better activity for promoting infection. The peptides synthesized in the experiment are P16-D, P16-K, P16-N, P16-2W and P16-W3K. for the synthesis of polypeptides. After testing the quality of MS and HPLC, the peptide can play the best effect on the enhancement of infection according to P16, that is, after infection of 4H in serum-free conditions, and then supplemented by serum or replaced as a complete medium, we have studied the infection promoting activity of the synthesized P16 derived peptide. Two factors which consider the efficiency of the infection and the cytotoxicity are considered. P16-D (Ac-671NW FAITNWLWYIK683K-KK-NH2) is chosen as the research object.
The results of virus infection test showed that the multiplier of P16-D and P16 enhanced virus infection were 13.9 and 7.5 respectively. It fully indicated that the reformed P16-D increased the infection activity by nearly 100% on the basis of P16. Compared with the optimum concentration DEAE and Polybrene, the cell survival rate in the infection process was 90% and 80%, respectively, and the toxicity of P16-D to the target cells. Sex is lower.
TEM, Tht specific binding experiments and EGCG experiments show that P16-D can form typical amyloid fibrils through low speed agitation and can effectively capture target cells and virus particles. Meanwhile, the virus particles can reach the concentrated target.P16-D amyloid fibrous activity against the envelope disease other than HIV-1 through a simple low-speed centrifugation. The virus - influenza virus and the FIV carrier system are also applicable.
The P16-D amyloid peptide can also effectively capture influenza virus particles; when P16-D exists, the blood coagulation titer of influenza virus is 4 times higher than that of the non polypeptide group; after influenza virus infection MDCK cells, the amount of virus in the supernatant is detected. The peptide group is obviously higher than the non peptide group, which indicates that the P16-D amyloid polypeptide will be immune to influenza virus immunity. A special role.
The ideal virus antigen should be immune to both body fluid immunity and cell immunity. Considering that P16-D fiber can effectively combine influenza virus particles and promote the infection of influenza virus at the cell level, we choose the reduced dose of influenza virus to be used together with the P16-D starch like fiber and inoculate mice through the nasal cavity. After that, the level of specific IgG antibody was lower than that in the normal dose virus group, but the level of the type antibody IgG1 was higher than that of the latter, and the level of the IgG2a was equal to that of the latter, indicating that the reduced dose of the attenuated influenza virus and the use of the P16-D amyloid polypeptide could also stimulate the organism to produce an effective immune response.
In conclusion, the superior infection activity of P16-D indicates that it will have a broad application prospect in retroviral infection and gene therapy. At the same time, its application in attenuated influenza immunity not only helps people to optimize the immune scheme, reduces the cost of production, but also promotes a deep understanding of the polypolymer of starch like fiber.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R392.11

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7 張e

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