本文選題：人生長(zhǎng)激素 + 枯草芽孢桿菌　； 參考：《江蘇大學(xué)》2014年博士論文

【摘要】：人生長(zhǎng)激素(human growth hormone,hGH)是一種重要的非糖基化蛋白質(zhì)激素。它包含有191個(gè)氨基酸分子,由人腦垂體中的生長(zhǎng)激素細(xì)胞進(jìn)行合成、存儲(chǔ)和分泌,是人類出生后促生長(zhǎng)的最主要激素和體內(nèi)代謝途徑的最重要調(diào)節(jié)因子。hGH生理功能主要包括：促進(jìn)軟骨、骨和組織的生長(zhǎng)；調(diào)節(jié)代謝作用,促進(jìn)脂肪分解和蛋白質(zhì)合成；調(diào)節(jié)機(jī)體免疫,維持機(jī)體的正常運(yùn)轉(zhuǎn)。天然人生長(zhǎng)激素非常稀少,因此,采用基因工程方法大規(guī)模生產(chǎn)hGH對(duì)滿足臨床大量需求具有重要的意義。目前,臨床上使用的hGH主要是采用以E.coli為宿主通過(guò)重組DNA技術(shù)制造的生長(zhǎng)激素(簡(jiǎn)稱rhGH)。然而,用大腸桿菌表達(dá)易形成包涵體,且需要通過(guò)變性、復(fù)性操作,工藝復(fù)雜,活性低,導(dǎo)致商品化產(chǎn)品rhGH產(chǎn)品成本非常昂貴。故而rhGH在醫(yī)學(xué)上雖然用途前景很廣,但當(dāng)前仍無(wú)法大范圍推廣應(yīng)用。 枯草芽孢桿菌是一種革蘭氏陽(yáng)性非致病性的益生菌,當(dāng)它處于營(yíng)養(yǎng)缺乏或其它環(huán)境脅迫的不良條件下能形成抗逆性的休眠體—芽孢。因?yàn)檠挎呔哂袕?qiáng)烈的抗逆性,能夠在極端的溫度、輻射和毒性化學(xué)物質(zhì)等不利的環(huán)境下長(zhǎng)期存活。因此,芽孢成為在極端環(huán)境中運(yùn)載外源蛋白或具有活性的生物大分子的有效載體。本研究的目的是通過(guò)枯草芽孢表面展示技術(shù)將hGH展示在芽孢表面,構(gòu)建具有營(yíng)養(yǎng)功能的表面展示hGH蛋白的重組芽孢。本研究的主要內(nèi)容和結(jié)果如下： (1)以攜帶人生長(zhǎng)激素hgh基因的載體為模板和PCR擴(kuò)增出人生長(zhǎng)激素基因hgh的編碼序列,并進(jìn)行測(cè)序和數(shù)據(jù)庫(kù)比對(duì)鑒定。 (2)將hgh克隆到質(zhì)粒pET30a上,使hgh基因前端連接腸激酶位點(diǎn)基因,構(gòu)建中間橋梁質(zhì)粒pET30a-hGH。 (3)將帶有腸激酶位點(diǎn)hgh基因克隆到質(zhì)粒pJS700上,使其與枯草芽孢桿菌芽孢衣殼蛋白基因cotC融合,構(gòu)建融合表達(dá)CotC-hGH的整合型重組質(zhì)粒pJS700-hGH。 (4)用質(zhì)粒pJS700-hGH轉(zhuǎn)化轉(zhuǎn)化枯草芽孢桿菌感受態(tài)細(xì)胞,篩選得到重組菌株并誘導(dǎo)該菌株產(chǎn)生芽孢,Western blot和免疫熒光實(shí)驗(yàn)進(jìn)一步鑒定hGH成功的展示在芽孢表面。 (5)用芽孢表面展示hGH的重組芽孢口添五齡家蠶六天后,分析家蠶的生理生化指標(biāo),結(jié)果發(fā)現(xiàn),口添芽孢的家蠶體重變化、蛹體重、體重轉(zhuǎn)換效率等顯著優(yōu)于對(duì)照。進(jìn)一步取家蠶血液進(jìn)行Western blot實(shí)驗(yàn),證實(shí)家蠶體內(nèi)產(chǎn)生的CotC-hGH融合蛋白被精確切除并加工成小肽hGH,并且hGH蛋白可以被消化吸收到家蠶血淋巴。 (6)進(jìn)一步用重組芽孢表面展示hGH的芽孢小鼠口服實(shí)驗(yàn),持續(xù)灌胃42天,摘眼球取血分析口服重組芽孢對(duì)小鼠血清生長(zhǎng)激素含量及小鼠血清生化常規(guī)指標(biāo)的影響。結(jié)果表明低劑量口服重組芽孢不會(huì)給小鼠胃腸道帶來(lái)不適,但是口服大劑量重組芽孢會(huì)影響小鼠各項(xiàng)生理指標(biāo)。 研究表明,利用枯草芽孢桿菌表面展示生物活性的hGH為當(dāng)前的人生長(zhǎng)激素制造開(kāi)辟了新途徑,同時(shí)為研究和應(yīng)用hGH的藥品和保健品奠定良好的基礎(chǔ)。
[Abstract]:Human growth hormone growth hormonehGH is an important nonglycosylated protein hormone. It contains 191 amino acid molecules synthesized, stored and secreted by growth hormone cells in the human pituitary gland. The physiological functions of HGH, which is the most important hormone and metabolic pathway in human body after birth, mainly include: promoting the growth of cartilage, bone and tissue, regulating metabolism, promoting fat decomposition and protein synthesis, promoting the growth of cartilage, bone and tissue, regulating metabolism, promoting fat catabolism and protein synthesis, promoting the growth of cartilage, bone and tissue. Regulate the immune system and maintain the normal operation of the body. Natural human growth hormone is very rare, so it is very important to produce hGH in large scale by genetic engineering. At present, the clinical use of hGH is mainly the use of E.coli as the host through recombinant DNA technology to produce growth hormone (rhGH). However, the expression of E. coli is easy to form inclusion bodies, and it needs denaturation, renaturation, complex process and low activity, which leads to the high cost of commercial rhGH products. Therefore, although rhGH is widely used in medicine, it can not be widely used at present. Bacillus subtilis (Bacillus subtilis) is a gram-positive, nonpathogenic probiotic bacterium. Because of its strong resistance to stress, spores can survive in adverse environments such as extreme temperatures, radiation and toxic chemicals. Therefore, spores have become an effective carrier for carrying foreign proteins or active biomolecules in extreme environments. The purpose of this study was to display hGH on the surface of spores by subtilis spores surface display technique, and construct recombinant spores with nutritional function to display hGH protein on the surface of spores. The main contents and results of this study are as follows: The encoding sequence of human growth hormone gene (hgh) was amplified by using the vector carrying human growth hormone (hgh) gene as template and PCR, and identified by sequencing and database alignment. (2) hgh was cloned into plasmid pET30a, and the front end of the hgh gene was connected with the enterokinase gene, and the intermediate bridge plasmid pET30a-hGH was constructed. The hgh gene with enterokinase site was cloned into plasmid pJS700 and fused with cotC of Bacillus subtilis spore capsid protein gene to construct an integrated recombinant plasmid pJS700-hGH expressing CotC-hGH. (4) plasmid pJS700-hGH was used to transform Bacillus subtilis into the competent cells, and the recombinant strain was screened and induced to produce spore by Western blot and immunofluorescence assay. Further, hGH was successfully displayed on the surface of the spores. The physiological and biochemical indexes of silkworm (Bombyx mori) were analyzed. The results showed that the body weight of silkworm, pupae weight and body weight conversion efficiency of silkworm with hGH were significantly better than that of control. The further Western blot test of silkworm blood confirmed that the CotC-hGH fusion protein produced in silkworm was accurately removed and processed into a small peptide hGHand the hGH protein could be digested and absorbed into the hemolymph of silkworm Bombyx mori. (6) the oral test of hGH spore mice was performed on the surface of recombinant spores. After 42 days of gavage, the effects of oral recombinant spores on serum growth hormone content and biochemical parameters of mice were analyzed. The results showed that low dose of recombinant spores did not bring discomfort to the gastrointestinal tract of mice, but oral administration of high dose of recombinant spores affected the physiological indexes of mice. The results showed that hGH, which showed bioactivity on the surface of Bacillus subtilis, opened up a new way for the production of human growth hormone, and laid a good foundation for the research and application of hGH drugs and health products.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R977.1

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