本文關(guān)鍵詞：重組人粒細(xì)胞集落刺激因子的長效性改造　出處：《山東師范大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

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【摘要】： 人粒細(xì)胞集落刺激因子(human granulocyte colony stimulating factor, hG-CSF)是一種定向作用于粒系祖細(xì)胞的造血生長因子。它作用于粒系祖細(xì)胞,可以特異刺激祖細(xì)胞向中性粒細(xì)胞增殖分化,并維持其功能和存活[1]。臨床上主要用于骨髓移植時(shí)促進(jìn)中性白細(xì)胞增加、癌癥化療引起的嚴(yán)重中性粒細(xì)胞缺乏癥。但是G-CSF的半衰期短,臨床治療應(yīng)用時(shí)需反復(fù)使用才能維持有效的血藥濃度,這導(dǎo)致病人的治療費(fèi)用高,病人的依從性差。本課題的目的是利用融合蛋白的方法,構(gòu)建具有更長半衰期的新型G-CSF,以取代普通的G-CSF。 根據(jù)文獻(xiàn)報(bào)道,有一種從噬菌體肽庫中篩選出來十肽能夠特異結(jié)合人IgG的Fc段(IgG Fc)。將編碼此小肽的基因與人G-CSF基因融合融合,構(gòu)建rhG-CSF融合蛋白(rhG-CSF-tag1)基因。 首先,設(shè)計(jì)引物,以rhG-CSF表達(dá)載體pBV-G-CSF為模板,進(jìn)行PCR反應(yīng),擴(kuò)增rhG-CSF編碼區(qū),及C-末端融合有多肽基因rhG-CSF融合蛋白(rhG-CSF-tag1)基因。將PCR產(chǎn)物以及空載體pBV220分別用EcoRI和BamHI雙酶切,然后,將酶切產(chǎn)物連接構(gòu)建出原核表達(dá)載體pBV-G-CSF和rhG-CSF-tag1。經(jīng)雙酶切鑒定、PCR鑒定及序列測定,表明兩個(gè)重組質(zhì)粒中的目的基因序列正確。 將陽性重組質(zhì)粒轉(zhuǎn)化大腸桿菌表達(dá)菌DH5α,在30℃條件下培養(yǎng)rhG-CSF和rhG-CSF-tag1工程菌, 42℃溫度誘導(dǎo)目的蛋白的表達(dá),并對(duì)目的蛋白在菌體內(nèi)的表達(dá)形式進(jìn)行分析。SDS-PAGE結(jié)果顯示,與對(duì)照菌相比,溫度誘導(dǎo)后的重組菌在分子量為19～21kDa處有目的蛋白表達(dá)。進(jìn)一步實(shí)驗(yàn)分析表明,在當(dāng)OD600達(dá)到0.4～0.6時(shí),升溫至42℃溫度誘導(dǎo)表達(dá)4h,目的蛋白質(zhì)的表達(dá)量最大,并且表達(dá)的目的蛋白主要以包涵體形式存在。 將rhG-CSF和rhG-CSF-tag1包涵體用各種不同的緩沖液進(jìn)行洗滌,可以去除一些雜蛋白,然后用尿素和鹽酸胍裂解包涵體,4℃透析復(fù)性,復(fù)性后的目標(biāo)蛋白用DEAE陰離子交換柱進(jìn)行純化,收集目的蛋白質(zhì)洗脫峰,得到純化的目的蛋白。將目的蛋白用Tris-HCl (pH8.0)緩沖液透析,之后換為HAc-NaAc(pH4.0)緩沖液繼續(xù)透析。 用G-CSF依賴株細(xì)胞株NFS-60細(xì)胞進(jìn)行rhG-CSF和rhG-CSF-tag1的體外活性測定。MTT法檢測表明,rhG-CSF和rhG-CSFtag1均能維持NFS-60細(xì)胞的存活與增殖。 用牛血清白蛋白作為標(biāo)準(zhǔn)蛋白繪制標(biāo)準(zhǔn)蛋白曲線,測出rhG-CSF和rhG-CSFtag1的蛋白濃度。最后,采用6～8周齡Balb/C小鼠,建立化療后白細(xì)胞低下小鼠模型,構(gòu)建成功后,分為陰性對(duì)照組、rhG-CSF治療組和rhG-CSFtag1組,分別注射10mM醋酸鹽緩沖液、rhG-CSF和rhG-CSFtag1。然后,每天對(duì)各組小鼠的白細(xì)胞進(jìn)行計(jì)數(shù)。 結(jié)果成功構(gòu)建了rhG-CSF和rhG-CSF-tag1表達(dá)載體,實(shí)現(xiàn)了其在大腸桿菌中高效表達(dá)。體外活性測定表明,復(fù)性后的rhG-CSF和rhG-CSF-tag1均能有效刺激G-CSF依賴細(xì)胞株NFS-60細(xì)胞的存活與增殖。動(dòng)物實(shí)驗(yàn)表明,融合蛋白rhG-CSF-tag1具有比rhG-CSF更長的體內(nèi)活性,這表明,rhG-CSF-tag1在體內(nèi)具有比rhG-CSF更長的半衰期。
[Abstract]:Human granulocyte colony stimulating factor (human granulocyte colony stimulating factor, hG-CSF) is a hematopoietic directional role in myeloid progenitor cell growth factor. Its role in myeloid progenitor cells can specifically stimulate progenitor cell differentiation to neutrophil cell proliferation, and maintain its power to promote the increase of neutrophils and survival [1]. mainly used for clinical bone marrow transplantation, cancer chemotherapy induced severe neutropenia. But G-CSF's short half-life, clinical application should be used repeatedly to maintain effective blood concentration, which led to the patient's high cost of treatment, patient compliance is poor. The purpose of this study is to use the method of fusion protein the construction of new, G-CSF has a longer half-life, to replace the ordinary G-CSF.
According to the literature, a Fc peptide (IgG Fc) that specifically binds human IgG is screened from phage peptide library. IgG fusion gene is fused with human G-CSF gene to construct rhG-CSF fusion protein (rhG-CSF-tag1) gene.
First of all, primers were designed with rhG-CSF expression vector pBV-G-CSF as template, PCR reaction to amplify the rhG-CSF encoding region, and C- terminal fusion peptide gene rhG-CSF fusion protein (rhG-CSF-tag1) gene. The product of PCR and plasmid pBV220 respectively with EcoRI and BamHI double enzyme digestion, then the digestion was connected to construct prokaryotic the expression vector pBV-G-CSF and rhG-CSF-tag1. through double enzyme digestion identification and sequence determination of PCR gene, showed that two recombinant plasmid in the correct sequence.
The positive recombinant plasmid was transformed into E.coli DH5 alpha, rhG-CSF and rhG-CSF-tag1 engineering bacteria cultivation under the condition of 30 DEG C, the temperature of 42 DEG C to induce the expression of protein, and the protein expression in the form of bacteria in vivo.SDS-PAGE analysis results showed that compared with the control strain, temperature induced recombinant bacteria in molecular weight 19 ~ 21kDa is the target protein expression. Further experiments showed that when the OD600 reaches 0.4 ~ 0.6 when heated to 42 DEG C temperature induced expression of 4h, expression level of target protein, and expression of target protein mainly in the form of inclusion bodies.
The buffer rhG-CSF and rhG-CSF-tag1 inclusion bodies with a variety of different washing, you can remove some miscellaneous protein, then using guanidine hydrochloride and urea inclusion crack unpacking 4 DEG C, renaturation, complex target protein after the DEAE anion exchange column for purification, the purpose of collecting the protein elution peak, purified protein. The target protein with Tris-HCl (pH8.0) bufferdialysis, then change to HAc-NaAc (pH4.0) buffer to dialysis.
The activity of rhG-CSF and rhG-CSF-tag1 in vitro was determined by G-CSF dependent cell line NFS-60 cells..MTT assay showed that both rhG-CSF and rhG-CSFtag1 could maintain NFS-60 cell survival and proliferation.
Using bovine serum albumin as the standard protein curve drawing standard protein, protein concentration measured rhG-CSF and rhG-CSFtag1. Finally, the 6~8 week old Balb/C mice leukopenia mice model after chemotherapy was constructed successfully, divided into negative control group, rhG-CSF group and rhG-CSFtag1 group were injected with 10mM acetate buffer, rhG-CSF and rhG-CSFtag1. then, the day of mice white blood cell count.
Results rhG-CSF was successfully constructed and rhG-CSF-tag1 expression vector, to achieve its high expression in Escherichia coli. The in vitro activity determination showed that the refolded rhG-CSF and rhG-CSF-tag1 can effectively stimulate G-CSF dependent survival and proliferation of NFS-60 cells. Animal experiments showed that the fusion protein of rhG-CSF-tag1 is longer than the rhG-CSF in this activity. Show that rhG-CSF-tag1 in vivo has a longer half-life than rhG-CSF.

【學(xué)位授予單位】：山東師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R346;Q789

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 付瑤;人粒細(xì)胞集落刺激因子的基因合成、原核表達(dá)與活性研究[D];吉林大學(xué);2011年

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本文編號(hào)：1375611