本文選題：穿膜肽-增強(qiáng)型綠色熒光蛋白 + 蛋白微球�。� 參考：《廣西醫(yī)科大學(xué)》2011年碩士論文

【摘要】：目的:探討應(yīng)用SPDP蛋白交聯(lián)法制備TAT-EGFP(穿膜肽-增強(qiáng)型綠色熒光蛋白)-抗體(抗HBsAg-IgG)-蛋白微球(BSA-NS)三聯(lián)偶聯(lián)物(TAT-EGFP-IgG-BSA-NS)的可行性,并鑒定該三聯(lián)偶聯(lián)物的化學(xué)成分、偶聯(lián)方式及穿膜活性;為后續(xù)構(gòu)建對腫瘤細(xì)胞的殺傷效應(yīng)同時具備安全性、靶向性、特異性和高效性的穿膜肽-腫瘤特異性抗體-藥物微球(包裹藥物的白蛋白微球)新型藥物傳送系統(tǒng)奠定基礎(chǔ)。 方法:1.大量表達(dá)融合蛋白TAT-EGFP并進(jìn)行鑒定:參照本課題組前期實(shí)驗(yàn)方法,復(fù)蘇實(shí)驗(yàn)室保存的高表達(dá)融合蛋白TAT-EGFP的表達(dá)菌株——大腸桿菌BL21,提取質(zhì)粒,進(jìn)行基因序列測定驗(yàn)證其目的質(zhì)粒的序列組成;大量誘導(dǎo)表達(dá)融合蛋白TAT-EGFP,并用鎳親和層析法進(jìn)行蛋白純化,用SDS-聚丙烯酰胺凝膠電泳方法(SDS-PAGE)鑒定其純度及分子量大小;用標(biāo)準(zhǔn)蛋白BCA法測定TAT-EGFP的濃度;將TAT-EGFP與EJ細(xì)胞(膀胱癌細(xì)胞系)共孵育,熒光顯微鏡下觀察融合蛋白TAT-EGFP的穿細(xì)胞膜活性。2.制備穿膜肽-蛋白微球二聯(lián)偶聯(lián)物:用異型雙功能交聯(lián)劑N-羥基琥珀酰亞胺基-3-(2-吡基二硫)-丙酸酯(SPDP)通過化學(xué)交聯(lián)的方法制備TAT-EGFP與蛋白微球(BSA-NS)的二聯(lián)偶聯(lián)物TAT-EGFP-BSA-NS;用SDS-PAGE法檢測二聯(lián)偶聯(lián)物的化學(xué)連接方式及二聯(lián)偶聯(lián)物成分;將二聯(lián)偶聯(lián)物與EJ細(xì)胞共孵育,熒光顯微鏡下觀察二聯(lián)偶聯(lián)物的穿細(xì)胞膜活性。3.制備穿膜肽-抗體-蛋白微球三聯(lián)偶聯(lián)物:用與2相同的方法制備TAT-EGFP、抗HBSAg-IgG與蛋白微球( BSA-NS )的三聯(lián)偶聯(lián)物TAT-EGFP-IgG-BSA-NS;4.TAT-EGFP、抗體與蛋白微球三聯(lián)偶聯(lián)物的鑒定:用SDS-PAGE法檢測三聯(lián)偶聯(lián)物的連接方式及化學(xué)組成;用拉曼光鑷顯微鏡系統(tǒng)(拉曼光鑷技術(shù))鑒定三聯(lián)偶聯(lián)物的化學(xué)成分;用非變性聚丙烯酰胺凝膠電泳(Native-PAGE)進(jìn)一步鑒定三聯(lián)偶聯(lián)物的蛋白微球及TAT-EGFP成分;用間接免疫熒光法進(jìn)一步鑒定三聯(lián)偶聯(lián)物中的抗體成分;將三聯(lián)偶聯(lián)物與EJ細(xì)胞共孵育,熒光顯微鏡下觀察三聯(lián)偶聯(lián)物的穿細(xì)胞膜活性。 結(jié)果:1.融合蛋白TAT-EGFP的表達(dá)與鑒定結(jié)果:序列測定結(jié)果顯示擴(kuò)增的TAT-EGFP基因與預(yù)期目的序列一致; SDS-PAGE可見純化后的蛋白無明顯雜帶,分子量大小約為31kd左右;標(biāo)準(zhǔn)蛋白BCA法測定的TAT-EGFP的濃度為14.2mg/ml;在激發(fā)波長為450nm-490nm的熒光顯微鏡下,可見與融合蛋白TAT-EGFP共孵育1小時的EJ細(xì)胞內(nèi)有綠色熒光顯示。2.二聯(lián)偶聯(lián)物的制備與鑒定結(jié)果:還原型SDS-PAGE電泳可見在低分子量端形成一條蛋白條帶,非還原型SDS-PAGE電泳未見條帶;在激發(fā)波長為450nm-490nm的熒光顯微鏡下,可以看到與二聯(lián)偶聯(lián)物共孵育1小時的EJ細(xì)胞內(nèi)有綠色熒光顯示。3.三聯(lián)偶聯(lián)物的制備及鑒定結(jié)果:還原型SDS-PAGE電泳可見在低分子量端形成三條蛋白條帶,非還原型SDS-PAGE電泳未見條帶;非變性聚丙烯酰胺凝膠在紫外凝膠成像儀下,可見加樣孔底部有白色顯示且未跑進(jìn)分離膠;光鑷?yán)庾V顯示三聯(lián)偶聯(lián)物較蛋白微球有一些峰變化,并且這些峰在抗體和TAT-EGFP純品的拉曼光譜上能找到對應(yīng)的峰;不同激發(fā)波長的熒光顯微鏡下,均可見微球表面分別發(fā)出綠色熒光和紅色熒光;在激發(fā)波長為450nm-490nm的熒光顯微鏡下,可以看到與三聯(lián)偶聯(lián)物共孵育1小時的EJ細(xì)胞內(nèi)有綠色熒光顯示。 結(jié)論:成功應(yīng)用SPDP蛋白交聯(lián)法制備穿膜肽-抗體-蛋白微球三聯(lián)偶聯(lián)物TAT-EGFP-IgG-BSA-NS,并證明三聯(lián)偶聯(lián)物具有穿膜活性;為后續(xù)構(gòu)建穿膜肽-腫瘤特異性抗體-藥物微球的新型偶聯(lián)藥物傳送系統(tǒng)奠定了基礎(chǔ)。 確定了用SPDP蛋白交聯(lián)法以抗體作為連接載體,將穿膜肽和蛋白微球連到抗體上構(gòu)建穿膜肽-抗體-蛋白微球三聯(lián)偶聯(lián)物(TAT-EGFP-IgG-BSA-NS)是可行的、較為理想的連接方案。首次應(yīng)用拉曼光鑷顯微鏡系統(tǒng)鑒定蛋白偶聯(lián)物的成分,其結(jié)果與SDS-PAGE法、非變性聚丙烯酰胺凝膠電泳方法、間接免疫熒光法等經(jīng)典方法鑒定結(jié)果一致,確定了拉曼光鑷顯微鏡系統(tǒng)進(jìn)行蛋白偶聯(lián)物的鑒定是可行的。
[Abstract]:Objective: To investigate the feasibility of using SPDP protein crosslinking method to prepare TAT-EGFP (transmembrane peptide - enhanced green fluorescent protein) - antibody (anti HBsAg-IgG) - protein microsphere (BSA-NS) triad coupling (TAT-EGFP-IgG-BSA-NS), and to identify the chemical composition, coupling method and membrane activity of the triad coupling, and to construct a subsequent killing effect on tumor cells. At the same time, it has the foundation of safety, targeting, specificity and high efficiency of membrane peptide - tumor specific antibody drug microspheres (encapsulated albumin microspheres) new drug delivery system.
Methods: 1. a large amount of fusion protein TAT-EGFP was expressed and identified: according to the preliminary experimental method of this group, Escherichia coli BL21, an expression strain of high expression fusion protein TAT-EGFP, preserved in the resuscitation laboratory, was extracted, and the sequence composition of the target plasmid was tested by gene sequencing, and a large amount of expression of fusion protein TAT-EGFP was induced. The purity and molecular weight of the protein were identified by SDS- polyacrylamide gel electrophoresis (SDS-PAGE), the concentration of TAT-EGFP was determined by standard protein BCA, and the TAT-EGFP and EJ cells (bladder cancer cell lines) were incubated, and the membrane active.2. of the fusion protein TAT-EGFP was prepared under the fluorescence microscope. Membrane peptide protein microsphere two coupling conjugate: preparation of TAT-EGFP and protein microsphere (BSA-NS) two coupling agent TAT-EGFP-BSA-NS by chemical crosslinking method using N- hydroxy succinimide -3- (2- pyyl two sulfur) propionate (SPDP), a heteromorphic double functional cross-linking agent, and the chemical connection and two coupling of two coupling agents by SDS-PAGE method Components; reincubating two couplet coupling with EJ cells and observing the membrane activity of the two couplet cell membrane under fluorescence microscope to prepare the membrane peptide antibody protein microsphere coupling compound: TAT-EGFP, HBSAg-IgG and protein microsphere (BSA-NS) triple coupling TAT-EGFP-IgG-BSA-NS, 4.TAT-EGFP, antibody and protein, were prepared in the same way as 2. Identification of triple coupling of microspheres: SDS-PAGE method was used to detect the connection and chemical composition of the triad coupling; the chemical constituents of the coupling were identified by Raman optical tweezers (Raman optical tweezers), and the protein microspheres and TAT-EGFP components of the coupling compounds were identified by Native-PAGE. The antibody components in the triplet coupling were further identified by indirect immunofluorescence, and the triplet coupling was incubated with EJ cells, and the membrane activity of the triplet coupling was observed under the fluorescence microscope.
Results: 1. fusion protein TAT-EGFP expression and identification results: sequencing results showed that the amplified TAT-EGFP gene was consistent with the intended purpose sequence; SDS-PAGE showed that the purified protein had no obvious stray bands, the molecular weight was about 31kd, and the concentration of TAT-EGFP measured by the standard protein BCA method was 14.2mg/ml; the excitation wavelength was 450nm-490nm. Under the fluorescence microscope, the results of the preparation and identification of the green fluorescent display.2. two coupling were found in the EJ cells which were incubated with the fusion protein TAT-EGFP for 1 hours. The reduced SDS-PAGE electrophoresis showed that a strip of protein was formed at the low molecular weight end, and no strip was found in the non prototype SDS-PAGE electrophoresis; the fluorescence microscopy at the excitation wavelength of 450nm-490nm was found. Under the microscope, we can see the preparation and identification results of the green fluorescent display.3. triple coupling in EJ cells which were incubated with the two coupling conjugate for 1 hours. The reduced SDS-PAGE electrophoresis showed that three protein bands were formed at the low molecular weight end, and no strip was found in the non prototype SDS-PAGE electrophoresis; the non denatured polyacrylamide gel was under the UV Gel imaging apparatus. It can be seen that there is a white display on the bottom of the sample hole and the separation glue is not running. The Raman spectra of the optical tweezers show that the coupling compounds have some peak changes over the protein microspheres, and these peaks can find the corresponding peaks on the Raman spectra of the antibodies and TAT-EGFP pure products. The green fluorescence of the microspheres is shown to be green fluorescence respectively under the fluorescence microscopy of different excitation wavelengths. Under the excitation wavelength of 450nm-490nm, a green fluorescent display was observed in EJ cells incubated for 1 hours with triplex conjugates.
Conclusion: SPDP protein crosslinking method was successfully used to prepare the membrane peptide antibody protein microsphere triple coupling agent TAT-EGFP-IgG-BSA-NS, and it was proved that the triplet coupling has the membrane activity, which laid the foundation for the subsequent construction of a new coupling drug delivery system for the membrane peptide - tumor specific antibody drug microspheres.
The SPDP protein crosslinking method was used to use the antibody as the connection carrier. The membrane peptide and the protein microspheres were linked to the antibody to construct the membrane peptide antibody protein microsphere triple coupling (TAT-EGFP-IgG-BSA-NS). It was a feasible and ideal connection scheme. The Raman optical tweezers microscope system was first used to identify the components of the protein coupling, and the results were with SDS-. The results of PAGE, non denatured polyacrylamide gel electrophoresis and indirect immunofluorescence are consistent. It is feasible to identify the protein coupling in the Raman tweezers microscope system.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R392

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 聶振;穿膜肽促進(jìn)β-葡萄糖苷酶/苦杏仁苷酶解前藥系統(tǒng)殺傷膀胱癌細(xì)胞的初步研究[D];廣西醫(yī)科大學(xué);2013年

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本文編號：1957187