鸡干扰素α和γ在家蚕中的表达及其抗病毒活性检测

发布时间：2018-05-07 02:34

本文选题：鸡干扰素α + 鸡干扰素γ　；参考：《中国农业科学院》2015年硕士论文

【摘要】：随着养鸡业的发展,鸡类疾病的防治越来越引起人们的重视。干扰素是一类多功能的细胞因子,在抗病毒、肿瘤和寄生虫方面都有良好的使用效果,对各种养鸡业常见的疾病,如新城疫、禽流感等都有良好的治疗作用。因此,开发高活性的鸡干扰素制品用于鸡类疾病的防治具有良好的应用前景。本研究选取Gen Bank数据库中的Ch IFN-α(Gen Bank:NM_205427.1)和Ch IFN-γ(Gen Bank:NM_205149.1)基因序列,按家蚕密码子偏好性对其进行优化。利用Optimum Gene TM方法,将Ch IFN-α基因和Ch IFN-γ基因的CAI值分别提高到0.88和0.89,调整Ch IFN-α基因和Ch IFN-γ的GC含量分别为49.13%和42.79%。同时,替换掉序列中的稀有密码子和能使m RNA产生发夹结构的密码子,并去除原始序列中的常用限制性内切酶酶切位点,然后采取化学合成法合成用于表达的完整的Ch IFN-α和Ch IFN-γ序列。将合成的Ch IFN-α和Ch IFN-γ序列插入杆状病毒基因组形成重组病毒,利用杆状病毒多基因表达技术在家蚕体内共表达Ch IFN-α和Ch IFN-γ。表达产物经Western blotting检测,证明Ch IFN-α和Ch IFN-γ成功在家蚕内表达。利用CEF/VSV-GFP系统检测表达产物的抗病毒活性,发现家蚕共表达的Ch IFN-α和Ch IFN-γ效价可达7.64×106U/m L。利用空斑筛选法筛选重组病毒,测得最高表达量的重组病毒共表达的Ch IFN-α和Ch IFN-γ的效价可达1.78×107U/m L,将表达量提高了约2.3倍。同时,检测了实验室利用杆状病毒单独表达的Ch IFN-α以及Ch IFN-γ的抗病毒活性。Ch IFN-α的抗病毒活性为3.66×106U/m L,Ch IFN-γ的抗病毒1.73×106U/m L。对家蚕表达的Ch IFN-α进行了初步纯化,结果表明经酸变性纯化后可初步去除掉蚕蛹中60k Da左右的杂蛋白,Ch IFN-α主要存在于变性后收获的上清中。本研究利用家蚕杆状病毒表达系统高效表达了Ch IFN-α和Ch IFN-γ,并检测到其具有较高的抗病毒活性,为干扰素制品的制备奠定了基础。
[Abstract]:With the development of chicken industry, people pay more and more attention to the prevention and treatment of chicken diseases. Interferon is a kind of multifunctional cytokines, which has good effect on antivirus, tumor and parasite, and has good therapeutic effect on common diseases in chicken industry, such as Newcastle disease, avian influenza and so on. Therefore, the development of high-activity chicken interferon products for the prevention and control of chicken diseases has a good application prospects. In this study, the Ch IFN- 伪 Gen Bank- Gen Bank-1) and Ch IFN- 纬 Gen Bank-: NM205149.1) gene sequences in Gen Bank database were selected and optimized according to the codon preference of silkworm, Bombyx mori (Bombyx mori). The CAI values of Ch IFN- 伪 gene and Ch IFN- 纬 gene were increased to 0.88 and 0.89 by Optimum Gene TM, respectively, and the GC contents of Ch IFN- 伪 gene and Ch IFN- 纬 were 49.13% and 42.79%, respectively. At the same time, the rare codon in the sequence and the codon that can cause m RNA to produce hairpin structure were replaced, and the common restriction endonuclease sites in the original sequence were removed. The complete Ch IFN- 伪 and Ch IFN- 纬 sequences were synthesized by chemical synthesis. The synthesized sequences of Ch IFN- 伪 and Ch IFN- 纬 were inserted into the genome of baculovirus to form recombinant virus and co-expressed Ch IFN- 伪 and Ch IFN- 纬 in silkworm by baculovirus polygene expression technique. Western blotting analysis showed that Ch IFN- 伪 and Ch IFN- 纬 were successfully expressed in Bombyx mori. The antiviral activity of the expressed product was detected by CEF/VSV-GFP system. It was found that the titers of Ch IFN- 伪 and Ch IFN- 纬 coexpressed in Bombyx mori could reach 7.64 脳 106U/m L. The recombinant virus was screened by plaque screening method. The titers of Ch IFN- 伪 and Ch IFN- 纬 coexpressed by the highest expression amount of recombinant virus were 1. 78 脳 107U/m / L, and the expression level was increased by about 2. 3 times. At the same time, the antiviral activity of Ch IFN- 伪 and Ch IFN- 纬 expressed by baculovirus alone in laboratory was determined. The antiviral activity of Ch IFN- 伪 was 3.66 脳 106U/m L Ch IFN- 纬, and the antivirus activity of Ch IFN- 纬 was 1.73 脳 106U/m L. The expression of Ch IFN- 伪 in silkworm, Bombyx mori, was preliminarily purified. The results showed that after acid denaturation and purification, about 60 kDa hetero protein Ch IFN- 伪 of silkworm pupae could be removed primarily in the supernatant harvested after denaturation. In this study, ChIFN- 伪 and Ch IFN- 纬 were highly expressed by the baculovirus expression system of Bombyx mori, and their antiviral activity was detected, which laid a foundation for the preparation of interferon products.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S859.79

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