本文選題：葡激酶 + 氨基酸特征結(jié)構(gòu)��； 參考：《重慶醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的通過對(duì)葡激酶(SAK)基因序列進(jìn)行定點(diǎn)突變、表達(dá)、純化與進(jìn)行聚乙二醇修飾以獲得較高純度的聚乙二醇化葡激酶(peg-SAK-cys),并對(duì)其溶栓活性與免疫原性初步進(jìn)行驗(yàn)證。方法設(shè)計(jì)引物將根據(jù)SAK晶體結(jié)構(gòu)與預(yù)測(cè)抗原位點(diǎn)所挑選的82號(hào)氨基酸突變?yōu)榘腚装彼岵⑼蛔冑|(zhì)粒通過化學(xué)轉(zhuǎn)化進(jìn)入BL21(DE3)感受態(tài)。利用經(jīng)典原核表達(dá)技術(shù)表達(dá)突變葡激酶蛋白(SAK-cys)。利用鎳離子交換柱、分子篩等方法分離純化SAK-cys蛋白并對(duì)其進(jìn)行聚乙二醇修飾。用纖維蛋白平板溶圈法和血栓彈力圖初步對(duì)本次實(shí)驗(yàn)制備的peg-SAK-cys與課題組以相同方法制備的另外4種peg-SAK-cys的生物活性進(jìn)行驗(yàn)證。以酶聯(lián)免疫吸附(ELISA)法評(píng)價(jià)這些peg-SAK-cys的免疫原性。分析體外實(shí)驗(yàn)數(shù)據(jù),并對(duì)功能評(píng)價(jià)較高的peg-SAK-cys通過動(dòng)物栓塞實(shí)驗(yàn)?zāi)Ｐ瓦M(jìn)一步功能驗(yàn)證。結(jié)果成功獲得了82號(hào)氨基酸突變的葡激酶質(zhì)粒,表達(dá)、純化了突變蛋白,并進(jìn)行聚乙二醇修飾獲得了第82號(hào)葡激酶突變的葡激酶蛋白(peg-SAK-C82A),分離純化后純度在總蛋白質(zhì)量的90%以上。統(tǒng)計(jì)分析與計(jì)算溶圈實(shí)驗(yàn)結(jié)果,第97號(hào)與104號(hào)氨基酸突變的聚乙二醇化葡激酶(peg-SAK-C97A、peg-SAK-C104G)保留了較高的活性;血栓彈力圖實(shí)驗(yàn)數(shù)據(jù)也支持了這一結(jié)果;免疫原性測(cè)定結(jié)果提示peg-SAK-C104G的免疫原性顯著低于野生型SAK(P=0.0002);評(píng)價(jià)peg-SAK-C104G對(duì)SD大鼠頸動(dòng)脈栓塞模型的溶栓效果,顯示蛋白于體內(nèi)實(shí)驗(yàn)也保留了相當(dāng)?shù)娜芩芰Α＝Y(jié)論通過位點(diǎn)特異性特變與聚乙二醇修飾技術(shù)的聯(lián)合運(yùn)用可以成功改造出有較低免疫原性的活性葡激酶。
[Abstract]:Objective to obtain high purity peg-SAK-cys1 by site-directed mutation, expression, purification and modification of staphylokinase SAK gene, and to verify its thrombolytic activity and immunogenicity. Methods A primer was designed to mutate the 82 amino acid selected according to the crystal structure and predicted antigenic site of SAK into cysteine and the mutant plasmid was chemically transformed into BL21DDE3). Classical prokaryotic expression technique was used to express the mutant staphylokinase protein SAK-cys1. SAK-cys protein was purified by nickel ion exchange column and molecular sieve and modified with polyethylene glycol. The bioactivity of peg-SAK-cys prepared in this experiment was preliminarily verified by fibrin plate method and thromboelastic diagram with the other four kinds of peg-SAK-cys prepared by the same method. Enzyme linked immunosorbent assay (Elisa) was used to evaluate the immunogenicity of these peg-SAK-cys. The experimental data in vitro were analyzed and the peg-SAK-cys with high function evaluation was further verified by animal embolization model. Results the staphylokinase plasmid with 82 amino acid mutation was successfully obtained, the mutant protein was expressed and purified, and the mutant protein peg-SAK-C82 was obtained by polyethylene glycol modification. The purity of the mutant protein was more than 90% of the total protein content. The results of statistical analysis and calculation showed that peg-SAK-C97Apeg-SAK-C104G) with amino acid mutations of 97 and 104 amino acids retained high activity, and the experimental data of thromboelastic diagram also supported the results. The results of immunogenicity test showed that the immunogenicity of peg-SAK-C104G was significantly lower than that of wild-type SAKP 0.0002, and the thrombolytic effect of peg-SAK-C104G on carotid artery embolization model of SD rats was evaluated, which showed that the protein also retained considerable thrombolytic ability in vivo. Conclusion the staphylokinase with low immunogenicity can be successfully modified by the combination of site-specific mutagenesis and polyethylene glycol modification.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R943

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相關(guān)期刊論文 前2條

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本文編號(hào)：1933602