钠泵α2亚基M4-M5区的克

发布时间：2018-08-29 10:49

【摘要】：Na~~+,K~~+-ATPase,又称钠泵,是高等真核生物细胞膜上普遍存在的一种特殊的膜结合蛋白,它在维持体内渗透压的平衡、稳定细胞膜电位、信号转导等方面具有重要作用。Na~~+,K~~+-ATPase是由α和β亚单位组成的异源二聚体,其中α亚单位是催化亚单位,它贯穿嵌入膜中,有10个跨膜区(M1～M10),具有酶的活性和许多钠泵调节因子的结合位点,其中M4～M5区直接参与了ATP的催化水解。钠泵α亚基有α1、α2、α3、α4四种亚型,各异构体间功能各异,具有种属特异性,近期的研究表明,钠泵α2亚基与高血压的发生和发展具有很大关系。本研究拟构建钠泵α2亚基M4～M5膜内区蛋白BB4-5的原核表达体系,表达并纯化目的蛋白,制备Na~+-K~+-ATPaseα2亚基的单克隆抗体,为进一步研究钠泵α2亚基在体内的作用机制,及与疾病发生的关系奠定基础。一、钠泵α2亚基M4-M5区蛋白的克隆和表达目的:利用基因重组技术构建钠泵α2亚基M4-M5膜内区蛋白的表达载体,IPTG诱导表达获得蛋白,通过蛋白纯化技术将蛋白纯化,为钠泵α2亚基单抗制备提供抗原。方法:(1)钠泵α2亚基M4-M5克隆载体的构建。以质粒YhNα2(含人钠泵α2亚基cDNA序列)为模板,PCR扩增得到α2亚基M4-M5区的基因片段BB4-5,将其加尾后连接至克隆载体pGM-T并转化大肠杆菌DH5α,挑选阳性克隆,得到质粒PGMT-BB4-5。(2)钠泵α2亚基M4-M5蛋白表达体系的构建。从质粒PGMT-BB4-5上切下目的基因片段,双酶切连接到表达载体pET28b(+)上并转化入大肠杆菌Rosetta2,挑选阳性克隆,得到质粒pET28b(+)-BB4-5。双酶切验证目的基因序列是否正确。1%接种表达菌,用终浓度为0.4 mM的IPTG进行诱导,SDS-PAGE电泳检测重组蛋白的表达。(3)重组蛋白的纯化:将菌体破碎,SDS-PAGE电泳分析目的蛋白存在的形式,将包涵体变性和复性后,用Ni-NTA亲和柱进行纯化,检测目的蛋白的纯度。(4)重组蛋白的Western blotting分析:将Ni2~+亲和层析纯化后的融合蛋白经SDS-PAGE蛋白电泳后,电转移到硝酸纤维素膜上,封闭,加入钠泵α2亚基抗血清或抗His抗体,洗膜,DAB显色试剂盒进行显色,凝胶成像仪照相。结果:(1)以质粒YhNα2为模板进行PCR扩增得到基因片段BB4-5,产物通过1%琼脂糖凝胶电泳检测,在相应的位置出现明显的条带。将其加尾后连接至pGM-T载体并转化入大肠杆菌DH5α,挑选阳性克隆扩增并提取质粒,双酶切和测序结果显示基因序列与理论一致。(2)将目的基因片段与同样双酶切的PET28b(+)表达载体相连,转化Rosetta2感受态细胞,在含Kan的平板上筛选重组子。双酶切结果表明蛋白表达体系构建成功。用IPTG诱导表达后,SDS-PAGE结果表明,BB4-5目的条带与理论值一致。(3)菌体破碎后,经SDS-PAGE电泳分析表明,表达产物主要以包涵体形式存在,将包涵体用盐酸胍变性,梯度稀释复性后,用Ni-NTA亲和柱进行纯化,得到纯度在90%以上的目的蛋白。(4)表达产物BB4-5经Western blotting分析,可与钠泵α2亚基的抗体及抗His抗体特异性结合。结论:本研究成功的构建了钠泵α2亚基M4-M5蛋白表达载体pET28b(+)-BB4-5,经IPTG诱导表达,Ni-NTA亲和柱进行纯化,得到纯度在90%以上的目的蛋白,可作为抗原来制备钠泵α2亚基单抗。二、钠泵α2亚基单抗的制备与鉴定目的:以钠泵α2亚基M4-M5膜内区重组蛋白为抗原,免疫BALB/c小鼠,通过杂交瘤技术制备单克隆抗体,并采用ELISA间接法和免疫印迹对单克隆抗体的特性进行和鉴定,为探明钠泵α2亚基在体内的作用机制,及其与疾病发生的关系奠定基础。方法:(1)将α2亚基纯化蛋白作为抗原,免疫6~8周BALB/c小鼠。(2)通过杂交瘤技术来筛选α2亚基的单克隆抗体:取被免疫动物的脾细胞(B淋巴细胞),与Sp2/0骨髓瘤细胞杂交;用间接ELISA法筛选阳性(抗体分泌)细胞;将阳性细胞通过有限稀释法进行克隆化,直接获得可稳定分泌单克隆抗体的杂交瘤细胞系,建株。(3)以小鼠腹水法制备杂交瘤的粗制单克隆抗体,采用辛酸-硫酸铵沉淀法将抗体进行纯化。(4)采用Sigma公司小鼠mAb Ig亚类检测试剂盒检测抗体的亚型。(5)采用Western-blotting法检测抗体的特异性。(6)参照Beatty的方法,采用间接ELISA法测定抗体的亲和常数。结果:(1)6~8周BALB/c小鼠免疫后,测得小鼠血清抗体效价均为1:50000以上。(2)选取抗体效价较高的小鼠分离脾细胞,采用常规方法与Sp2/0骨髓瘤细胞进行融合,通过多次间接ELISA实验进行筛选,检测出阳性孔有14孔,克隆化后得到两株效价较高细胞株。(3)小鼠腹腔接种杂交瘤细胞株,收集腹水,通过辛酸-硫酸铵沉淀法进行纯化,得到纯化的mAb。(4)通过小鼠mAb Ig亚类检测试剂盒检测两株mAb的亚型均为IgG2a。(5)Western-blotting结果显示两株mAb与钠泵M4-M5膜内区蛋白特异性结合。(6)测定两种细胞株分泌的mAb亲和常数分别为3.51×108L/ mol和5.88×107 L/ mol。结论:利用基因重组表达的钠泵M4-M5膜内区蛋白作为抗原,通过杂交瘤技术,成功制备两株单克隆抗体,并采用间接ELISA法和免疫印迹对其进行鉴定。该单抗的获得,为进一步探讨钠泵α2亚基在体内的作用机制,及其与疾病发生的关系奠定基础。
[Abstract]:Na~+, K~+-ATPase, also known as sodium pump, is a special membrane-binding protein ubiquitous in higher eukaryotic cell membranes. It plays an important role in maintaining the balance of osmotic pressure in vivo, stabilizing cell membrane potential, signal transduction and so on. Na~+, K~+-ATPase is a heterodimer composed of alpha and beta subunits, in which alpha subunits are catalysts. Sodium pump alpha subunits have four subtypes, alpha 1, alpha 2, alpha 3, and alpha 4, with different functions and species specificity. Recent studies have shown that sodium pump alpha 2 is involved in the catalytic hydrolysis of ATP. In this study, we intend to construct a prokaryotic expression system of sodium pump alpha2 subunit M4-M5 intramembrane protein BB4-5, express and purify the target protein, prepare monoclonal antibodies against Na + - K + - ATPase alpha2 subunit, and further study the mechanism of action of sodium pump alpha2 subunit in vivo and the relationship between sodium pump alpha2 subunit and disease occurrence. Lay the foundation.
1. Cloning and expression of M4-M5 region protein of sodium pump subunit alpha 2. Objective: To construct the expression vector of M4-M5 region protein of sodium pump subunit alpha 2 by gene recombination technique. The protein was obtained by IPTG induction. The protein was purified by protein purification technique to provide antigen for the preparation of sodium pump subunit alpha 2 monoclonal antibody.
METHODS: (1) Construction of the cloning vector of sodium pump alpha 2 subunit M4-M5. The gene fragment BB4-5 of the M4-M5 region of the alpha 2 subunit was amplified by PCR using plasmid YhNalpha2 (containing the cDNA sequence of human sodium pump alpha 2 subunit) as template. The gene fragment BB4-5 was tailed to the cloning vector pGM-T and transformed into E. coli DH5a. The positive clone was selected and the plasmid PGMT-BB4-5 was obtained. Construction of the white expression system. The target gene fragment was cut from the plasmid PGMT-BB4-5, linked to the expression vector pET28b (+) by double digestion and transformed into Escherichia coli Rosetta2. The plasmid pET28b (+) -BB4-5 was obtained by double digestion to verify the correct sequence of the target gene. 1% of the expressed strain was inoculated and induced by IPTG with the final concentration of 0.4 mM. SDS-PAGE electrophoresis was used to detect the expression of the recombinant protein. (3) Purification of the recombinant protein: fragmentation of the bacteria, SDS-PAGE analysis of the existence of the target protein, denaturation and renaturation of the inclusion body, purification of the inclusion body with Ni-NTA affinity column, detection of the purity of the target protein. (4) Western blotting analysis of the recombinant protein: purification of Ni2~+ affinity chromatography after melting. After SDS-PAGE protein electrophoresis, the synthase protein was transferred to the nitrocellulose membrane, blocked, added sodium pump alpha 2 subunit antiserum or anti-His antibody, washed, developed by DAB color reagent kit, and photographed by gel imager.
Results: (1) The gene fragment BB4-5 was amplified by PCR using plasmid YhNalpha2 as template, and the product was detected by 1% agarose gel electrophoresis. The product was connected to pGM-T vector and transformed into E. coli DH5alpha. The positive clones were selected and amplified, and the plasmid was extracted. The results of double enzyme digestion and sequencing showed that the gene sequence was sequenced. (2) The target gene fragment was linked to the same double-digested PET28b (+) expression vector, transformed Rosetta2 receptor cells, and screened recombinant plasmids on Kan-containing platforms. The results of double-digestion showed that the protein expression system was successfully constructed. After fragmentation, SDS-PAGE electrophoresis showed that the expressed product was mainly in the form of inclusion body. The inclusion body was denatured by guanidine hydrochloride and renatured by gradient dilution. The purified protein was purified by Ni-NTA affinity column. (4) The expression product BB4-5 was analyzed by Western blotting and could be used as an antibody against sodium pump alpha2 subunit and anti-Hist subunit. Body specific binding.
CONCLUSION: The expression vector pET28b (+) - BB4-5 of sodium pump subunit M4-M5 protein was successfully constructed and expressed by IPTG and purified by Ni-NTA affinity column.
Two, preparation and identification of sodium pump alpha 2 subunit monoclonal antibody
Objective: To immunize BALB/c mice with recombinant protein of intramembrane region of sodium pump alpha 2 subunit M4-M5 as antigen, prepare monoclonal antibody by hybridoma technique, and identify the characteristics of monoclonal antibody by indirect ELISA and immunoblotting, so as to explore the mechanism of action of sodium pump alpha 2 subunit in vivo and its relationship with disease.
Methods: (1) BALB/c mice were immunized with purified protein of alpha 2 subunit for 6-8 weeks. (2) Monoclonal antibodies against alpha 2 subunit were screened by hybridoma technique. Spleen cells (B lymphocytes) of immunized animals were hybridized with Sp2/0 myeloma cells, positive (antibody secretion) cells were screened by indirect ELISA, and positive cells were introduced by limited dilution method. The hybridoma cell lines secreting monoclonal antibodies stably were cloned and constructed. (3) The crude monoclonal antibodies against hybridoma were prepared from ascites of mice and purified by octanoic acid-ammonium sulfate precipitation. (4) The subtypes of the antibodies were detected by Sigma mouse mAb Ig subclass detection kit. (5) Western-blotting. (6) The affinity constants of antibodies were determined by indirect ELISA with reference to Beatty's method.
Results: (1) After 6-8 weeks of immunization, the antibody titers of BALB/c mice were all above 1:50 000. (2) Sp2/0 myeloma cells were fused with normal method and screened by indirect ELISA. The positive pore was detected to be 14, and two strains with high titer were obtained after cloning. (3) Mice were inoculated with hybridoma cell lines, ascites were collected and purified by octanoic acid-ammonium sulfate precipitation method. The purified mAb. (4) Both mAb subtypes were detected to be IgG2a by mouse mAb Ig subclass detection kit. (5) Western-blotting results showed that the two mAb strains were specifically bound to intramembrane protein of sodium pump M4-M5. The mAb affinity constants of the two cell lines were 3.51 x 108L/ mol and 5.88 x 107 L/ mol., respectively.
CONCLUSION: Two monoclonal antibodies were successfully prepared by hybridoma technique and identified by indirect ELISA and Western blot, using recombinant M4-M5 intramembrane protein as antigen. Basics.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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