鸡CARP单克隆抗体制备及其在骨骼肌发育中的表达

发布时间：2018-01-02 18:27

本文关键词：鸡CARP单克隆抗体制备及其在骨骼肌发育中的表达　出处：《黑龙江八一农垦大学》2010年硕士论文　论文类型：学位论文

【摘要】： 心肌锚定重复蛋白(CARP)是一种重要的核转录调控因子,是锚定重复序列家族成员之一,具有锚定重复区、两个PEST基序以及一个核定位信号。目前CARP已经被确定为一个新的心血管疾病的分子标志物,它在胚胎期、新生期和成年哺乳动物心脏中高水平表达。CARP具有多种功能,在血管发生,心脏发育,细胞凋亡,转录调控,纤维的装配和拉伸传感,核质通讯等方面具有重要作用。最近研究发现与哺乳动物相比,CARP在鸡骨骼肌组织中呈特异性表达,这暗示了CARP可能在鸡骨骼肌发育过程起重要作用,因此深入研究CARP在鸡骨骼肌发生中的调控机制具有重要意义。为了研究CARP在鸡骨骼肌发育过程中的表达,本试验克隆表达并纯化了鸡CARP重组蛋白,并制备了抗鸡CARP单克隆抗体,在蛋白质水平来检测其在骨骼肌发育过程中的表达。利用RT-PCR的方法从鸡骨骼肌扩增获得CARP N端110个氨基酸的cDNA,将其克隆至原核表达载体pET28b中,然后转化至大肠杆菌BL21(DE3),提取质粒进行双酶切鉴定,并测序验证。重组菌经IPTG诱导表达并纯化重组蛋白。SDS-PAGE结果表明与未诱导菌株相比,诱导后的重组菌表达分子量约18000的重组蛋白,并主要以可溶性形式存在,该蛋白经灰度分析纯度能达到88.5%。用纯化的CARP重组蛋白免疫8周龄BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞融合,间接ELISA法进行筛选阳性克隆,经多次克隆化培养,筛选高分泌滴度的阳性杂交瘤细胞株,制备腹水并纯化。采用McAb亚类鉴定试剂盒确定单克隆抗体亚型,对筛选得到的杂交瘤细胞染色体分析,检测细胞培养上清和腹水效价,并用Western blot方法对McAbs进行特异性鉴定。为研究CARP在肌肉不同发育时期的表达,本实验选取胚胎期,出生后幼雏,成年鸡为研究对象,从鸡肌骨骼肌提取总蛋白,采用Western blot检测肌肉发育过程中CARP在蛋白质水平的表达。结果最终获得两株能稳定分泌抗鸡CARP杂交瘤细胞株,分命名为4A8、4E62,亚型都属于IgG1,轻链为κ链;融合细胞的染色体数目均大于89条,符合杂交瘤染色体数目。两株杂交瘤细胞免疫小鼠的腹水及其细胞培养液上清的效价分别为3.2×104、1.28×105和2.56×103、2.56×103。Western blot结果表明McAb能识别重组表达和内源性的CARP蛋白,并且与小鼠CARP蛋白无交叉反应。在蛋白质水平,CARP仅在出壳后的鸡骨骼肌中表达。本研究成功获得2株能识别鸡CARP的杂交瘤及其分泌的特异性McAbs,为进一步研究CARP的功能奠定了物质基础;试验结果表明其CARP可能在鸡骨骼肌发育过程中发挥重要作用。
[Abstract]:Cardiac Anchorage repeat protein (CARP) is an important nuclear transcription regulator and a member of the family of anchoring repeats with anchoring repeats. Two PEST motifs and a nuclear locus signal. CARP has been identified as a new molecular marker of cardiovascular disease at embryonic stage. High level expression of .CARP in newborn and adult mammalian hearts has many functions, such as angiogenesis, cardiac development, apoptosis, transcriptional regulation, fiber assembly and stretch sensing. Nuclear and cytoplasmic communication plays an important role. Recently, it has been found that Carp is specifically expressed in chicken skeletal muscle compared with mammalian. This suggests that CARP may play an important role in the development of chicken skeletal muscle. Therefore, it is of great significance to study the regulatory mechanism of CARP in chicken skeletal muscle. In order to study the expression of CARP during the development of chicken skeletal muscle. The recombinant chicken CARP protein was cloned and purified, and monoclonal antibody against chicken CARP was prepared to detect its expression in skeletal muscle development at protein level. CARP N-terminal 110 amino acid cDNA was amplified from chicken skeletal muscle by RT-PCR and cloned into prokaryotic expression vector pET28b. Then it was transformed into E. coli BL21 and DE3, and the plasmid was digested and identified by double enzyme digestion. The recombinant strain was induced by IPTG and purified by SDS-PAGE. After induction, the recombinant protein with molecular weight of about 18000 was expressed and mainly existed in soluble form. The purity of the protein reached 88.5 by gray level analysis. The purified CARP recombinant protein was used to immunize 8-week-old BALB/c mice. The spleen cells were fused with SP2/0 myeloma cells and the positive clones were screened by indirect ELISA method. The positive hybridoma cell lines with high secretory titers were screened after multiple cloning, ascites were prepared and purified. Monoclonal antibody subtypes were determined by McAb subclass identification kit. Chromosome analysis of the selected hybridoma cells was performed to detect the titer of cell culture supernatant and ascites. In order to study the expression of CARP in different stages of muscle development, the embryonic and postnatal chicks were selected to identify the specificity of McAbs by Western blot. The total protein was extracted from the skeletal muscle of the adult chicken and the expression of CARP in the muscle development was detected by Western blot. Results two hybridoma cell lines with stable secretion of chicken CARP were obtained, named as 4A8, 4E62.The subtypes belonged to IgG1 and the light chain was 魏 chain. The chromosome number of the fusion cells was more than 89, which was consistent with the number of hybridoma chromosomes. The titers of ascites and supernatants of the two hybridoma cells immunized mice were 3.2 脳 104, respectively. The results of 1.28 脳 10 ~ 5 and 2.56 脳 10 ~ (3) Western blot showed that McAb could recognize recombinant and endogenous CARP proteins. There was no cross reaction with mouse CARP protein and the protein level was only expressed in chicken skeletal muscle after exfoliation. In this study, two strains of hybridoma recognized chicken CARP and specific McAbs secreted were successfully obtained, which laid a material foundation for further study on the function of CARP. The results suggest that CARP may play an important role in the development of chicken skeletal muscle.
【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392.11

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