新型聚乙二醇化葡激酶的表達(dá)、純化及功能鑒定研究
發(fā)布時(shí)間：2018-11-02 12:06
【摘要】：目的通過(guò)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。方法根據(jù)SAK蛋白晶體結(jié)構(gòu)及抗原位點(diǎn)選擇突變位點(diǎn),設(shè)計(jì)引物將所選的氨基酸突變?yōu)榘腚装彼�。將突變質(zhì)粒通過(guò)化學(xué)轉(zhuǎn)化進(jìn)入BL21(DE3)感受態(tài),并利用經(jīng)典原核表達(dá)技術(shù),在大腸桿菌中表達(dá)突變葡激酶(SAK-cys)。利用鎳離子交換柱、分子篩等方法分離純化目的蛋白。用纖維蛋白平板溶圈法和血栓彈力圖初步對(duì)其生物活性進(jìn)行驗(yàn)證。以酶聯(lián)免疫吸附(ELISA)法評(píng)價(jià)peg-SAK-cys的免疫原性。結(jié)果成功獲得了SAK-cys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peg-SAK-cys,分離純化后純度在總蛋白質(zhì)量的90%以上。計(jì)算其溶圈實(shí)驗(yàn)結(jié)果,活性為8.2×10~4IU·mg~(-1);血栓彈力圖實(shí)驗(yàn)結(jié)果提示其具有較高的溶栓活性;免疫原性測(cè)定結(jié)果提示peg-SAK-cys免疫原性低于野生型SAK(P=0.000 2)。結(jié)論通過(guò)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SAK。
[Abstract]:Objective to obtain a high purity (SAK) gene by site-directed mutation, expression, purification and modification with polyethylene glycol (PEG). Its thrombolytic activity and immunogenicity were preliminarily verified. Methods according to the crystal structure and antigenic site of SAK protein mutation sites were selected and primers were designed to mutate the selected amino acids to cysteine. The mutant plasmid was chemically transformed into BL21 (DE3) competent state and expressed in E. coli by classical prokaryotic expression technique. The target protein was purified by nickel ion exchange column and molecular sieve. The bioactivity was preliminarily verified by fibrin plate method and thromboelastic diagram. The immunogenicity of peg-SAK-cys was evaluated by enzyme linked immunosorbent assay (ELISA). Results the SAK-cys plasmid was successfully obtained, the mutant protein was expressed and purified, and the purity of peg-SAK-cys, was more than 90% of the total protein. The results of circle dissolution test showed that the activity was 8.2 脳 10~4IU mg~ (-1), the thromboelastogram showed that the thrombolytic activity was higher, and the immunogenicity of peg-SAK-cys was lower than that of wild type SAK (P0. 0002). Conclusion SAK. with low immunogenicity can be successfully modified by the combination of site-specific mutagenesis and polyethylene glycol modification.
【作者單位】： 重慶醫(yī)科大學(xué)附屬第一醫(yī)院心內(nèi)科;重慶醫(yī)科大學(xué)檢驗(yàn)醫(yī)學(xué)院;
【基金】：重慶市科委資助項(xiàng)目(cstc2012gg-gjhz0025)
【分類(lèi)號(hào)】：R915
，
本文編號(hào)：2305962
[Abstract]:Objective to obtain a high purity (SAK) gene by site-directed mutation, expression, purification and modification with polyethylene glycol (PEG). Its thrombolytic activity and immunogenicity were preliminarily verified. Methods according to the crystal structure and antigenic site of SAK protein mutation sites were selected and primers were designed to mutate the selected amino acids to cysteine. The mutant plasmid was chemically transformed into BL21 (DE3) competent state and expressed in E. coli by classical prokaryotic expression technique. The target protein was purified by nickel ion exchange column and molecular sieve. The bioactivity was preliminarily verified by fibrin plate method and thromboelastic diagram. The immunogenicity of peg-SAK-cys was evaluated by enzyme linked immunosorbent assay (ELISA). Results the SAK-cys plasmid was successfully obtained, the mutant protein was expressed and purified, and the purity of peg-SAK-cys, was more than 90% of the total protein. The results of circle dissolution test showed that the activity was 8.2 脳 10~4IU mg~ (-1), the thromboelastogram showed that the thrombolytic activity was higher, and the immunogenicity of peg-SAK-cys was lower than that of wild type SAK (P0. 0002). Conclusion SAK. with low immunogenicity can be successfully modified by the combination of site-specific mutagenesis and polyethylene glycol modification.
【作者單位】： 重慶醫(yī)科大學(xué)附屬第一醫(yī)院心內(nèi)科;重慶醫(yī)科大學(xué)檢驗(yàn)醫(yī)學(xué)院;
【基金】：重慶市科委資助項(xiàng)目(cstc2012gg-gjhz0025)
【分類(lèi)號(hào)】：R915
，
本文編號(hào)：2305962
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