重组人粒细胞集落刺激因子的长效性改造

发布时间：2018-01-03 21:16

本文关键词：重组人粒细胞集落刺激因子的长效性改造　出处：《山东师范大学》2008年硕士论文　论文类型：学位论文

【摘要】： 人粒细胞集落刺激因子(human granulocyte colony stimulating factor, hG-CSF)是一种定向作用于粒系祖细胞的造血生长因子。它作用于粒系祖细胞,可以特异刺激祖细胞向中性粒细胞增殖分化,并维持其功能和存活[1]。临床上主要用于骨髓移植时促进中性白细胞增加、癌症化疗引起的严重中性粒细胞缺乏症。但是G-CSF的半衰期短,临床治疗应用时需反复使用才能维持有效的血药浓度,这导致病人的治疗费用高,病人的依从性差。本课题的目的是利用融合蛋白的方法,构建具有更长半衰期的新型G-CSF,以取代普通的G-CSF。根据文献报道,有一种从噬菌体肽库中筛选出来十肽能够特异结合人IgG的Fc段(IgG Fc)。将编码此小肽的基因与人G-CSF基因融合融合,构建rhG-CSF融合蛋白(rhG-CSF-tag1)基因。首先,设计引物,以rhG-CSF表达载体pBV-G-CSF为模板,进行PCR反应,扩增rhG-CSF编码区,及C-末端融合有多肽基因rhG-CSF融合蛋白(rhG-CSF-tag1)基因。将PCR产物以及空载体pBV220分别用EcoRI和BamHI双酶切,然后,将酶切产物连接构建出原核表达载体pBV-G-CSF和rhG-CSF-tag1。经双酶切鉴定、PCR鉴定及序列测定,表明两个重组质粒中的目的基因序列正确。将阳性重组质粒转化大肠杆菌表达菌DH5α,在30℃条件下培养rhG-CSF和rhG-CSF-tag1工程菌, 42℃温度诱导目的蛋白的表达,并对目的蛋白在菌体内的表达形式进行分析。SDS-PAGE结果显示,与对照菌相比,温度诱导后的重组菌在分子量为19～21kDa处有目的蛋白表达。进一步实验分析表明,在当OD600达到0.4～0.6时,升温至42℃温度诱导表达4h,目的蛋白质的表达量最大,并且表达的目的蛋白主要以包涵体形式存在。将rhG-CSF和rhG-CSF-tag1包涵体用各种不同的缓冲液进行洗涤,可以去除一些杂蛋白,然后用尿素和盐酸胍裂解包涵体,4℃透析复性,复性后的目标蛋白用DEAE阴离子交换柱进行纯化,收集目的蛋白质洗脱峰,得到纯化的目的蛋白。将目的蛋白用Tris-HCl (pH8.0)缓冲液透析,之后换为HAc-NaAc(pH4.0)缓冲液继续透析。用G-CSF依赖株细胞株NFS-60细胞进行rhG-CSF和rhG-CSF-tag1的体外活性测定。MTT法检测表明,rhG-CSF和rhG-CSFtag1均能维持NFS-60细胞的存活与增殖。用牛血清白蛋白作为标准蛋白绘制标准蛋白曲线,测出rhG-CSF和rhG-CSFtag1的蛋白浓度。最后,采用6～8周龄Balb/C小鼠,建立化疗后白细胞低下小鼠模型,构建成功后,分为阴性对照组、rhG-CSF治疗组和rhG-CSFtag1组,分别注射10mM醋酸盐缓冲液、rhG-CSF和rhG-CSFtag1。然后,每天对各组小鼠的白细胞进行计数。结果成功构建了rhG-CSF和rhG-CSF-tag1表达载体,实现了其在大肠杆菌中高效表达。体外活性测定表明,复性后的rhG-CSF和rhG-CSF-tag1均能有效刺激G-CSF依赖细胞株NFS-60细胞的存活与增殖。动物实验表明,融合蛋白rhG-CSF-tag1具有比rhG-CSF更长的体内活性,这表明,rhG-CSF-tag1在体内具有比rhG-CSF更长的半衰期。
[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.

【学位授予单位】：山东师范大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R346;Q789

【引证文献】