穿膜肽—抗体—蛋白微球偶联物的制备及鉴定

发布时间：2018-05-30 23:01

本文选题：穿膜肽-增强型绿色荧光蛋白 + 蛋白微球　；参考：《广西医科大学》2011年硕士论文

【摘要】：目的:探讨应用SPDP蛋白交联法制备TAT-EGFP(穿膜肽-增强型绿色荧光蛋白)-抗体(抗HBsAg-IgG)-蛋白微球(BSA-NS)三联偶联物(TAT-EGFP-IgG-BSA-NS)的可行性,并鉴定该三联偶联物的化学成分、偶联方式及穿膜活性;为后续构建对肿瘤细胞的杀伤效应同时具备安全性、靶向性、特异性和高效性的穿膜肽-肿瘤特异性抗体-药物微球(包裹药物的白蛋白微球)新型药物传送系统奠定基础。方法:1.大量表达融合蛋白TAT-EGFP并进行鉴定:参照本课题组前期实验方法,复苏实验室保存的高表达融合蛋白TAT-EGFP的表达菌株——大肠杆菌BL21,提取质粒,进行基因序列测定验证其目的质粒的序列组成;大量诱导表达融合蛋白TAT-EGFP,并用镍亲和层析法进行蛋白纯化,用SDS-聚丙烯酰胺凝胶电泳方法(SDS-PAGE)鉴定其纯度及分子量大小;用标准蛋白BCA法测定TAT-EGFP的浓度;将TAT-EGFP与EJ细胞(膀胱癌细胞系)共孵育,荧光显微镜下观察融合蛋白TAT-EGFP的穿细胞膜活性。2.制备穿膜肽-蛋白微球二联偶联物:用异型双功能交联剂N-羟基琥珀酰亚胺基-3-(2-吡基二硫)-丙酸酯(SPDP)通过化学交联的方法制备TAT-EGFP与蛋白微球(BSA-NS)的二联偶联物TAT-EGFP-BSA-NS;用SDS-PAGE法检测二联偶联物的化学连接方式及二联偶联物成分;将二联偶联物与EJ细胞共孵育,荧光显微镜下观察二联偶联物的穿细胞膜活性。3.制备穿膜肽-抗体-蛋白微球三联偶联物:用与2相同的方法制备TAT-EGFP、抗HBSAg-IgG与蛋白微球( BSA-NS )的三联偶联物TAT-EGFP-IgG-BSA-NS;4.TAT-EGFP、抗体与蛋白微球三联偶联物的鉴定:用SDS-PAGE法检测三联偶联物的连接方式及化学组成;用拉曼光镊显微镜系统(拉曼光镊技术)鉴定三联偶联物的化学成分;用非变性聚丙烯酰胺凝胶电泳(Native-PAGE)进一步鉴定三联偶联物的蛋白微球及TAT-EGFP成分;用间接免疫荧光法进一步鉴定三联偶联物中的抗体成分;将三联偶联物与EJ细胞共孵育,荧光显微镜下观察三联偶联物的穿细胞膜活性。结果:1.融合蛋白TAT-EGFP的表达与鉴定结果:序列测定结果显示扩增的TAT-EGFP基因与预期目的序列一致; SDS-PAGE可见纯化后的蛋白无明显杂带,分子量大小约为31kd左右;标准蛋白BCA法测定的TAT-EGFP的浓度为14.2mg/ml;在激发波长为450nm-490nm的荧光显微镜下,可见与融合蛋白TAT-EGFP共孵育1小时的EJ细胞内有绿色荧光显示。2.二联偶联物的制备与鉴定结果:还原型SDS-PAGE电泳可见在低分子量端形成一条蛋白条带,非还原型SDS-PAGE电泳未见条带;在激发波长为450nm-490nm的荧光显微镜下,可以看到与二联偶联物共孵育1小时的EJ细胞内有绿色荧光显示。3.三联偶联物的制备及鉴定结果:还原型SDS-PAGE电泳可见在低分子量端形成三条蛋白条带,非还原型SDS-PAGE电泳未见条带;非变性聚丙烯酰胺凝胶在紫外凝胶成像仪下,可见加样孔底部有白色显示且未跑进分离胶;光镊拉曼光谱显示三联偶联物较蛋白微球有一些峰变化,并且这些峰在抗体和TAT-EGFP纯品的拉曼光谱上能找到对应的峰;不同激发波长的荧光显微镜下,均可见微球表面分别发出绿色荧光和红色荧光;在激发波长为450nm-490nm的荧光显微镜下,可以看到与三联偶联物共孵育1小时的EJ细胞内有绿色荧光显示。结论:成功应用SPDP蛋白交联法制备穿膜肽-抗体-蛋白微球三联偶联物TAT-EGFP-IgG-BSA-NS,并证明三联偶联物具有穿膜活性;为后续构建穿膜肽-肿瘤特异性抗体-药物微球的新型偶联药物传送系统奠定了基础。确定了用SPDP蛋白交联法以抗体作为连接载体,将穿膜肽和蛋白微球连到抗体上构建穿膜肽-抗体-蛋白微球三联偶联物(TAT-EGFP-IgG-BSA-NS)是可行的、较为理想的连接方案。首次应用拉曼光镊显微镜系统鉴定蛋白偶联物的成分,其结果与SDS-PAGE法、非变性聚丙烯酰胺凝胶电泳方法、间接免疫荧光法等经典方法鉴定结果一致,确定了拉曼光镊显微镜系统进行蛋白偶联物的鉴定是可行的。
[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.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

【引证文献】