【摘要】：Na~~+,K~~+-ATPase,又稱鈉泵,是高等真核生物細(xì)胞膜上普遍存在的一種特殊的膜結(jié)合蛋白,它在維持體內(nèi)滲透壓的平衡、穩(wěn)定細(xì)胞膜電位、信號(hào)轉(zhuǎn)導(dǎo)等方面具有重要作用。Na~~+,K~~+-ATPase是由α和β亞單位組成的異源二聚體,其中α亞單位是催化亞單位,它貫穿嵌入膜中,有10個(gè)跨膜區(qū)(M1～M10),具有酶的活性和許多鈉泵調(diào)節(jié)因子的結(jié)合位點(diǎn),其中M4～M5區(qū)直接參與了ATP的催化水解。鈉泵α亞基有α1、α2、α3、α4四種亞型,各異構(gòu)體間功能各異,具有種屬特異性,近期的研究表明,鈉泵α2亞基與高血壓的發(fā)生和發(fā)展具有很大關(guān)系。本研究擬構(gòu)建鈉泵α2亞基M4～M5膜內(nèi)區(qū)蛋白BB4-5的原核表達(dá)體系,表達(dá)并純化目的蛋白,制備Na~+-K~+-ATPaseα2亞基的單克隆抗體,為進(jìn)一步研究鈉泵α2亞基在體內(nèi)的作用機(jī)制,及與疾病發(fā)生的關(guān)系奠定基礎(chǔ)。 一、鈉泵α2亞基M4-M5區(qū)蛋白的克隆和表達(dá)目的:利用基因重組技術(shù)構(gòu)建鈉泵α2亞基M4-M5膜內(nèi)區(qū)蛋白的表達(dá)載體,IPTG誘導(dǎo)表達(dá)獲得蛋白,通過(guò)蛋白純化技術(shù)將蛋白純化,為鈉泵α2亞基單抗制備提供抗原。 方法:(1)鈉泵α2亞基M4-M5克隆載體的構(gòu)建。以質(zhì)粒YhNα2(含人鈉泵α2亞基cDNA序列)為模板,PCR擴(kuò)增得到α2亞基M4-M5區(qū)的基因片段BB4-5,將其加尾后連接至克隆載體pGM-T并轉(zhuǎn)化大腸桿菌DH5α,挑選陽(yáng)性克隆,得到質(zhì)粒PGMT-BB4-5。(2)鈉泵α2亞基M4-M5蛋白表達(dá)體系的構(gòu)建。從質(zhì)粒PGMT-BB4-5上切下目的基因片段,雙酶切連接到表達(dá)載體pET28b(+)上并轉(zhuǎn)化入大腸桿菌Rosetta2,挑選陽(yáng)性克隆,得到質(zhì)粒pET28b(+)-BB4-5。雙酶切驗(yàn)證目的基因序列是否正確。1%接種表達(dá)菌,用終濃度為0.4 mM的IPTG進(jìn)行誘導(dǎo),SDS-PAGE電泳檢測(cè)重組蛋白的表達(dá)。(3)重組蛋白的純化:將菌體破碎,SDS-PAGE電泳分析目的蛋白存在的形式,將包涵體變性和復(fù)性后,用Ni-NTA親和柱進(jìn)行純化,檢測(cè)目的蛋白的純度。(4)重組蛋白的Western blotting分析:將Ni2~+親和層析純化后的融合蛋白經(jīng)SDS-PAGE蛋白電泳后,電轉(zhuǎn)移到硝酸纖維素膜上,封閉,加入鈉泵α2亞基抗血清或抗His抗體,洗膜,DAB顯色試劑盒進(jìn)行顯色,凝膠成像儀照相。 結(jié)果:(1)以質(zhì)粒YhNα2為模板進(jìn)行PCR擴(kuò)增得到基因片段BB4-5,產(chǎn)物通過(guò)1%瓊脂糖凝膠電泳檢測(cè),在相應(yīng)的位置出現(xiàn)明顯的條帶。將其加尾后連接至pGM-T載體并轉(zhuǎn)化入大腸桿菌DH5α,挑選陽(yáng)性克隆擴(kuò)增并提取質(zhì)粒,雙酶切和測(cè)序結(jié)果顯示基因序列與理論一致。(2)將目的基因片段與同樣雙酶切的PET28b(+)表達(dá)載體相連,轉(zhuǎn)化Rosetta2感受態(tài)細(xì)胞,在含Kan的平板上篩選重組子。雙酶切結(jié)果表明蛋白表達(dá)體系構(gòu)建成功。用IPTG誘導(dǎo)表達(dá)后,SDS-PAGE結(jié)果表明,BB4-5目的條帶與理論值一致。(3)菌體破碎后,經(jīng)SDS-PAGE電泳分析表明,表達(dá)產(chǎn)物主要以包涵體形式存在,將包涵體用鹽酸胍變性,梯度稀釋復(fù)性后,用Ni-NTA親和柱進(jìn)行純化,得到純度在90%以上的目的蛋白。(4)表達(dá)產(chǎn)物BB4-5經(jīng)Western blotting分析,可與鈉泵α2亞基的抗體及抗His抗體特異性結(jié)合。 結(jié)論:本研究成功的構(gòu)建了鈉泵α2亞基M4-M5蛋白表達(dá)載體pET28b(+)-BB4-5,經(jīng)IPTG誘導(dǎo)表達(dá),Ni-NTA親和柱進(jìn)行純化,得到純度在90%以上的目的蛋白,可作為抗原來(lái)制備鈉泵α2亞基單抗。 二、鈉泵α2亞基單抗的制備與鑒定 目的:以鈉泵α2亞基M4-M5膜內(nèi)區(qū)重組蛋白為抗原,免疫BALB/c小鼠,通過(guò)雜交瘤技術(shù)制備單克隆抗體,并采用ELISA間接法和免疫印跡對(duì)單克隆抗體的特性進(jìn)行和鑒定,為探明鈉泵α2亞基在體內(nèi)的作用機(jī)制,及其與疾病發(fā)生的關(guān)系奠定基礎(chǔ)。 方法:(1)將α2亞基純化蛋白作為抗原,免疫6~8周BALB/c小鼠。(2)通過(guò)雜交瘤技術(shù)來(lái)篩選α2亞基的單克隆抗體:取被免疫動(dòng)物的脾細(xì)胞(B淋巴細(xì)胞),與Sp2/0骨髓瘤細(xì)胞雜交;用間接ELISA法篩選陽(yáng)性(抗體分泌)細(xì)胞;將陽(yáng)性細(xì)胞通過(guò)有限稀釋法進(jìn)行克隆化,直接獲得可穩(wěn)定分泌單克隆抗體的雜交瘤細(xì)胞系,建株。(3)以小鼠腹水法制備雜交瘤的粗制單克隆抗體,采用辛酸-硫酸銨沉淀法將抗體進(jìn)行純化。(4)采用Sigma公司小鼠mAb Ig亞類檢測(cè)試劑盒檢測(cè)抗體的亞型。(5)采用Western-blotting法檢測(cè)抗體的特異性。(6)參照Beatty的方法,采用間接ELISA法測(cè)定抗體的親和常數(shù)。 結(jié)果:(1)6~8周BALB/c小鼠免疫后,測(cè)得小鼠血清抗體效價(jià)均為1:50000以上。(2)選取抗體效價(jià)較高的小鼠分離脾細(xì)胞,采用常規(guī)方法與Sp2/0骨髓瘤細(xì)胞進(jìn)行融合,通過(guò)多次間接ELISA實(shí)驗(yàn)進(jìn)行篩選,檢測(cè)出陽(yáng)性孔有14孔,克隆化后得到兩株效價(jià)較高細(xì)胞株。(3)小鼠腹腔接種雜交瘤細(xì)胞株,收集腹水,通過(guò)辛酸-硫酸銨沉淀法進(jìn)行純化,得到純化的mAb。(4)通過(guò)小鼠mAb Ig亞類檢測(cè)試劑盒檢測(cè)兩株mAb的亞型均為IgG2a。(5)Western-blotting結(jié)果顯示兩株mAb與鈉泵M4-M5膜內(nèi)區(qū)蛋白特異性結(jié)合。(6)測(cè)定兩種細(xì)胞株分泌的mAb親和常數(shù)分別為3.51×108L/ mol和5.88×107 L/ mol。 結(jié)論:利用基因重組表達(dá)的鈉泵M4-M5膜內(nèi)區(qū)蛋白作為抗原,通過(guò)雜交瘤技術(shù),成功制備兩株單克隆抗體,并采用間接ELISA法和免疫印跡對(duì)其進(jìn)行鑒定。該單抗的獲得,為進(jìn)一步探討鈉泵α2亞基在體內(nèi)的作用機(jī)制,及其與疾病發(fā)生的關(guān)系奠定基礎(chǔ)。
[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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R363

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3 張s，

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