P16-D淀粉样多肽促进逆转录病毒感染以及在流感免疫中的应用

发布时间：2018-05-18 19:43

本文选题：基因转导 + P16衍生肽　；参考：《吉林大学》2014年硕士论文

【摘要】：提高基因治疗的效率需要合适的基因载体系统和逐渐发展的用于增强基因转导化合物的使用。逆转录病毒载体使外源基因在宿主细胞内实现稳定、长期的表达而占有很大的优势。病毒与靶细胞表面的负电荷产生的静电斥力大大限制了基因转导的效率。用于增强基因转导的化合物从最初的阳离子化合物Poly-L-lysine等发展至今，商品化的DEAE dextran和polybrene仍被人们广泛应用于基因转导的实验中。最近的研究表明，淀粉样纤维多肽在促进基因转导过程中具有突出的优势，如SEVI和EF-C，已经被人们证实可以提高逆转录病毒基因转导的效率，而且显著强于DEAE和polybrene。最近我们发现合成的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末端增加三个赖氨酸，也能够形成淀粉样纤维结构促进HIV-1感染，并且效率强于SEVI。对P13和P16促感染机理的研究表明，芳香族氨基酸色氨酸和碱性氨基酸赖氨酸对P16的促感染活性很重要。由它们的作用方式来看，淀粉样纤维和正电性是多肽多聚物行使功能的必要条件。本文的研究主要是在有利于淀粉样纤维结构形成的前提下，对P16序列中的酸性氨基酸--天冬氨酸进行突变或增加序列中碱性氨基酸的数目，以期获得促感染活性更好的多肽。实验中合成的多肽有P16-D、P16-K、P16-N、P16-2W和P16-W3K。对合成的多肽进行MS和HPLC的质量检测无误后，按照P16发挥最佳感染增强效果的条件，即在无血清条件下感染4h后再补加血清或更换为完全培养基，我们对合成的P16衍生肽的促感染活性进行了研究。综合考虑促进病毒感染的效率和细胞毒性两个因素，选用P16-D（Ac-671NW FAITNWLWYIK683K-KK-NH2）为研究对象。病毒感染实验结果表明P16-D与P16增强病毒感染的倍数分别是13.9和7.5，充分说明改造后的P16-D的促感染活性在P16基础上提高将近100%，同时与最适作用浓度DEAE和polybrene相比，促感染过程中细胞存活率分别是90%与80%左右，P16-D对靶细胞的毒性更低。 TEM、Tht特异性结合实验及EGCG实验表明P16-D经过低速搅拌形成典型的淀粉样纤维结构，能够有效地捕获靶细胞和病毒颗粒，同时也使病毒颗粒通过简单地低速离心就可以达到浓缩的目的。P16-D淀粉样纤维的促感染活性对HIV-1以外的包膜病毒—流感病毒及FIV载体系统也适用。鉴于P16-D淀粉样多肽也能有效地捕获流感病毒颗粒；在P16-D存在时，流感病毒的血凝效价比不加多肽组提高4倍；流感病毒感染MDCK细胞后，检测上清里的病毒量，加肽组明显高于不加肽组，预示着P16-D淀粉样多肽将在流感病毒免疫中发挥特殊的作用。理想的病毒抗原经免疫后应该既能引起体液免疫，又能引起细胞免疫。考虑到P16-D纤维能够有效地结合流感病毒颗粒，在细胞水平上促进流感病毒的感染，我们选择降低剂量的流感病毒配合P16-D淀粉样纤维一起使用后，经鼻腔接种小鼠后，特异性IgG抗体水平低于正常剂量病毒组，但是分型抗体IgG1的水平比后者要高，IgG2a的水平则与之相当，，表明降低剂量的减毒流感病毒配合P16-D淀粉样多肽使用，也能够刺激机体产生有效的免疫应答。综上，P16-D优越的促感染活性预示着它将在逆转录病毒感染和基因治疗中具有广阔的应用前景，同时，它在减毒的流感免疫中的应用不仅帮助人们优化免疫方案，降低生产成本，而且推动了对淀粉样纤维多聚物的深入认识。
[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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R392.11

【共引文献】