本文選題：雞干擾素α + 雞干擾素γ��； 參考：《中國(guó)農(nóng)業(yè)科學(xué)院》2015年碩士論文

【摘要】：隨著養(yǎng)雞業(yè)的發(fā)展,雞類(lèi)疾病的防治越來(lái)越引起人們的重視。干擾素是一類(lèi)多功能的細(xì)胞因子,在抗病毒、腫瘤和寄生蟲(chóng)方面都有良好的使用效果,對(duì)各種養(yǎng)雞業(yè)常見(jiàn)的疾病,如新城疫、禽流感等都有良好的治療作用。因此,開(kāi)發(fā)高活性的雞干擾素制品用于雞類(lèi)疾病的防治具有良好的應(yīng)用前景。本研究選取Gen Bank數(shù)據(jù)庫(kù)中的Ch IFN-α(Gen Bank:NM_205427.1)和Ch IFN-γ(Gen Bank:NM_205149.1)基因序列,按家蠶密碼子偏好性對(duì)其進(jìn)行優(yōu)化。利用Optimum Gene TM方法,將Ch IFN-α基因和Ch IFN-γ基因的CAI值分別提高到0.88和0.89,調(diào)整Ch IFN-α基因和Ch IFN-γ的GC含量分別為49.13%和42.79%。同時(shí),替換掉序列中的稀有密碼子和能使m RNA產(chǎn)生發(fā)夾結(jié)構(gòu)的密碼子,并去除原始序列中的常用限制性內(nèi)切酶酶切位點(diǎn),然后采取化學(xué)合成法合成用于表達(dá)的完整的Ch IFN-α和Ch IFN-γ序列。將合成的Ch IFN-α和Ch IFN-γ序列插入桿狀病毒基因組形成重組病毒,利用桿狀病毒多基因表達(dá)技術(shù)在家蠶體內(nèi)共表達(dá)Ch IFN-α和Ch IFN-γ。表達(dá)產(chǎn)物經(jīng)Western blotting檢測(cè),證明Ch IFN-α和Ch IFN-γ成功在家蠶內(nèi)表達(dá)。利用CEF/VSV-GFP系統(tǒng)檢測(cè)表達(dá)產(chǎn)物的抗病毒活性,發(fā)現(xiàn)家蠶共表達(dá)的Ch IFN-α和Ch IFN-γ效價(jià)可達(dá)7.64×106U/m L。利用空斑篩選法篩選重組病毒,測(cè)得最高表達(dá)量的重組病毒共表達(dá)的Ch IFN-α和Ch IFN-γ的效價(jià)可達(dá)1.78×107U/m L,將表達(dá)量提高了約2.3倍。同時(shí),檢測(cè)了實(shí)驗(yàn)室利用桿狀病毒單獨(dú)表達(dá)的Ch IFN-α以及Ch IFN-γ的抗病毒活性。Ch IFN-α的抗病毒活性為3.66×106U/m L,Ch IFN-γ的抗病毒1.73×106U/m L。對(duì)家蠶表達(dá)的Ch IFN-α進(jìn)行了初步純化,結(jié)果表明經(jīng)酸變性純化后可初步去除掉蠶蛹中60k Da左右的雜蛋白,Ch IFN-α主要存在于變性后收獲的上清中。本研究利用家蠶桿狀病毒表達(dá)系統(tǒng)高效表達(dá)了Ch IFN-α和Ch IFN-γ,并檢測(cè)到其具有較高的抗病毒活性,為干擾素制品的制備奠定了基礎(chǔ)。
[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- 偽 Gen Bank- Gen Bank-1) and Ch IFN- 緯 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- 偽 gene and Ch IFN- 緯 gene were increased to 0.88 and 0.89 by Optimum Gene TM, respectively, and the GC contents of Ch IFN- 偽 gene and Ch IFN- 緯 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- 偽 and Ch IFN- 緯 sequences were synthesized by chemical synthesis. The synthesized sequences of Ch IFN- 偽 and Ch IFN- 緯 were inserted into the genome of baculovirus to form recombinant virus and co-expressed Ch IFN- 偽 and Ch IFN- 緯 in silkworm by baculovirus polygene expression technique. Western blotting analysis showed that Ch IFN- 偽 and Ch IFN- 緯 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- 偽 and Ch IFN- 緯 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- 偽 and Ch IFN- 緯 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- 偽 and Ch IFN- 緯 expressed by baculovirus alone in laboratory was determined. The antiviral activity of Ch IFN- 偽 was 3.66 脳 106U/m L Ch IFN- 緯, and the antivirus activity of Ch IFN- 緯 was 1.73 脳 106U/m L. The expression of Ch IFN- 偽 in silkworm, Bombyx mori, was preliminarily purified. The results showed that after acid denaturation and purification, about 60 kDa hetero protein Ch IFN- 偽 of silkworm pupae could be removed primarily in the supernatant harvested after denaturation. In this study, ChIFN- 偽 and Ch IFN- 緯 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.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S859.79

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 李宇彩;基因工程干擾素的純化研究[J];國(guó)外醫(yī)學(xué).預(yù)防.診斷.治療用生物制品分冊(cè);1996年01期

2 呂志慧;高瑾;劉振勇;鄭世民;;雞干擾素的抗球蟲(chóng)作用及應(yīng)用前景[J];黑龍江畜牧獸醫(yī);2007年06期

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本文編號(hào)：1855030