靶向黑色素瘤重组免疫毒素MSH-PE38KDEL的表达、纯化及其靶向抗肿瘤生物学效应
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摘要
恶性黑色素瘤是恶性肿瘤中发病率增长最快的一种，目前仍没有找到治疗转移性黑色素瘤最为有效的方法。重组毒素由于具有特异性高、细胞毒性强的特点，有望成为黑色素瘤（辅助）治疗的有效方法之一，特别是对转移性黑色素瘤的治疗可能是最好的方式。本研究利用恶性黑色素瘤细胞表面MC1R的结合位点为正常细胞及其它肿瘤的几十倍的巨大差异，并利用促黑色素细胞激素(Melanophore-stimulating hormone, α-MSH)可特异性的与恶性黑色素瘤表面的MC1R结合的特性，将α-MSH作为恶性黑色素瘤特异的靶向载体；将绿脓杆菌外毒素PE作为免疫毒素的毒性部分用于杀伤靶细胞，构建了重组毒素MSH-PE38KDEL，以基因工程方式表达黑色素瘤靶向免疫毒素。
重组毒素MSH-PE38KDEL的构建与表达。本研究通过生物信息学分析，优化MSH与PE40的链接Linker，确保各自的结构与功能不受影响；然后对PE40进行修饰，降低其免疫原性（PE38），并增加其活性（PE38KDEL）；构建了MSH-PE38KDEL基因，将其连接到pET28a、pHis1525表达载体上，构建了pET28a-MSH-PE38KDEL、 pHis1525-MSH-PE38KDEL重组质粒，分别在Rosegami、WH320表达宿主菌内进行了表达，经SDS-PAGE分析及活性测定，最终筛选了高表达且具有生物活性的重组质粒pET28a-MSH-PE38KDEL。将序列鉴定正确的重组质粒转化入Rosetta-gamiTM2(DE3)，IPTG诱导目的蛋白表达。以SDS-PAGE对表达产物进行鉴定。同时用凝胶成像分析系统配套软件对MSH-PE38KDEL融合蛋白的表达量进行分析，证明其表达量可占全菌体的10%。
重组毒素MSH-PE38KDEL的纯化。利用Western blot对超声破碎后菌体进行分析证明融合蛋白为可溶性表达。对表达后蛋白采用了菌体超声破碎、离心、疏水层析、离子交换及分子筛层析等纯化手段， SDS-PAGE分析MSH-PE38KDEL的纯度，其纯度可达90%以上。
重组毒素MSH-PE38KDEL的体外抗黑色素瘤活性。采用α-MSH受体高表达的人黑色素瘤A875细胞及鼠黑色素瘤B16细胞进行了体外生物活性试验，同时以α-MSH受体低表达的鼻咽癌细胞CNE、乳腺癌细胞MCF及人正常细胞2BS做对照，证明其靶向杀伤作用。MTT检测MSH-PE38KDEL对黑色素瘤细胞A875及B16的杀伤作用在85%以上，而对人正常细胞2BS无杀伤作用。
重组毒素MSH-PE38KDEL的体内抗黑色素瘤活性。以鼠黑色素瘤B16细胞在NIH/nu裸鼠体内建立黑色素瘤高转移动物模型。采用了瘤周围注射及静脉注射两种给药途径，对荷瘤小鼠进行治疗。结果显示以MSH-PE38KDEL1.1mg/只连续注射10天，可显著抑制肿瘤生长，其抑瘤率＞90%，且瘤周注射组肿瘤消失率为30%，静脉注射组肿瘤消失率为40%。病理切片HE染色结果显示MSH-PE38KDEL有效抑制了黑色素瘤B16细胞的肝内转移；电镜观察肝脏超微结构结果显示，MSH-PE38KDEL抑制了黑色素瘤B16细胞对机体细胞的破坏作用。重组毒素MSH-PE38KDEL在体内具有明显的抗黑色素瘤的效应，毒副作用低，病理及电镜结果显示对实质脏器均未见损伤。MSH-PE38KDEL有望成为靶向治疗黑色素瘤的候选药物。
Malignant melanoma is one of the malignant tumors with the fastest incidencegrowth. So far, the most effective therapy to the metastatic melanoma has not beendiscovered. Recombinant toxin is expected to become one of the most effective(assisting) therapy to melanoma for its high particularity and strong cytotoxity,especially to the metastatic melanoma. This study concentrated on the specificcombination of α-MSH with MC1R on the surface of malignant melanoma, as well asthe dozens of times’ gap between the binding site of MC1R on the surface ofmalignant melanoma and that of normal cells and other tumors, to make it as aspecific targeting carrier of malignant melanoma. The study also used pseudomonasexotoxin (PE) as the toxic part of immunotoxin, to build a recombinant toxin called asMSH-PE38KDEL, expressing melanoma targeting toxin in the way of geneticengineering.
Construction and expression of recombinant toxin—MSH-PE38KDEL. In this study,the linker of MSH and PE40was optimized by bioinformatics analysis, to ensure thattheir relative structure and function were not to be affected. Then PE40was modified,to reduce its immunogenicity (PE38) and increase its activity (PE38KDEL). Next thegene (MSH-PE38KDEL) was constructed and linked to the expression vectors—pET28a and pHis1525, to construct two recombinant plasmids—pET28a(+)-MSH-PE38KDEL and pHis1525-MSH-PE38KDEL, and they were expressed in theexpression host—Rosetta-gamiTM2(DE3) and WH320. By SDS-PAGE analysis andactivity determination, the recombinant plasmid with high expression andtoxicity—pET28a(+)-MSH-PE38KDEL was finally screened out. The plasmid wastranslated into Rosetta-gamiTM2(DE3) cell after it was determined with the correctsequence, and IPTG was used to induce fusion protein MSH-PE38KDEL expression,with SDS-PAGE as the determination on the expression products. Meanwhile, thesupporting software of gel image analysis system was applied to the analysis on expression level of MSH-PE38KDEL fusion protein, which took10%of the wholebacteria
Purification of recombinant toxin MSH-PE38KDEL. The Western Blot was usedto make somatic analyze after ultrasonic disruption to prove the soluble expression offusion protein. Purification methods such as somatic ultrasonic disruption,centrifugation, hydrophobic chromatography, ion exchange, molecular sievechromatography were adopted on the protein after expression. The purity quotient ofMSH-PE38KDEL with SDS-PAGE obtained over90%.
The in vitro antimelanoma activity of recombinant toxin MSH-PE38KDEL.The study tested the in vitro biological activity of human melanoma A875cells andmurine melanoma B16cells with high expression of α-MSH receptor, comparingthem with CNE, MCF, and2BS cells, whose α-MSH receptor expressions are low, inorder to prove its targeted killing effect. MTT test results showed that the killingeffect of MSH-PE38KDEL on melanoma cells A875and B16was over85%, and0onthe human normal2BS cells.
In vivo antimelanoma activity of recombinant toxin MSH-PE38KDEL. Themurine melanoma B16-cell was adopted in nude mice to establish a melanoma highlymetastatic animal model. The tumor-bearing mice were administrated with the routesof peri-tumor injection and intravenous injection. The result was that after10consecutive days of daily injection with the MSH-PE38KDEL concentration of1.1mg/mouse, the tumor growth was inhibited significantly with the rate of over90%.Meanwhile, the rate of disappearance in the group of peri-tumor injection was30%,and in the group of intravenous injection—that was40%. The HE staining resultshowed that MSH-PE38KDEL effectively inhibited the metastasis of melanoma B16cells in livers. The ultra structure of liver electron microscopy revealed thatMSH-PE38KDEL inhibited the damaging effects of melanoma B16cells on the livercells. Recombinant toxin MSH-PE38KDEL owned obvious antimelanoma effects invivo, with low toxic and side effects. The pathology results and electron microscopyrevealed no damage to the parenchymatous. In all, MSH-PE38KDEL is expected tobe adopted as one of the drug options to the targeting treatment for malignantmelanoma.
重组毒素MSH-PE38KDEL的构建与表达。本研究通过生物信息学分析，优化MSH与PE40的链接Linker，确保各自的结构与功能不受影响；然后对PE40进行修饰，降低其免疫原性（PE38），并增加其活性（PE38KDEL）；构建了MSH-PE38KDEL基因，将其连接到pET28a、pHis1525表达载体上，构建了pET28a-MSH-PE38KDEL、 pHis1525-MSH-PE38KDEL重组质粒，分别在Rosegami、WH320表达宿主菌内进行了表达，经SDS-PAGE分析及活性测定，最终筛选了高表达且具有生物活性的重组质粒pET28a-MSH-PE38KDEL。将序列鉴定正确的重组质粒转化入Rosetta-gamiTM2(DE3)，IPTG诱导目的蛋白表达。以SDS-PAGE对表达产物进行鉴定。同时用凝胶成像分析系统配套软件对MSH-PE38KDEL融合蛋白的表达量进行分析，证明其表达量可占全菌体的10%。
重组毒素MSH-PE38KDEL的纯化。利用Western blot对超声破碎后菌体进行分析证明融合蛋白为可溶性表达。对表达后蛋白采用了菌体超声破碎、离心、疏水层析、离子交换及分子筛层析等纯化手段， SDS-PAGE分析MSH-PE38KDEL的纯度，其纯度可达90%以上。
重组毒素MSH-PE38KDEL的体外抗黑色素瘤活性。采用α-MSH受体高表达的人黑色素瘤A875细胞及鼠黑色素瘤B16细胞进行了体外生物活性试验，同时以α-MSH受体低表达的鼻咽癌细胞CNE、乳腺癌细胞MCF及人正常细胞2BS做对照，证明其靶向杀伤作用。MTT检测MSH-PE38KDEL对黑色素瘤细胞A875及B16的杀伤作用在85%以上，而对人正常细胞2BS无杀伤作用。
重组毒素MSH-PE38KDEL的体内抗黑色素瘤活性。以鼠黑色素瘤B16细胞在NIH/nu裸鼠体内建立黑色素瘤高转移动物模型。采用了瘤周围注射及静脉注射两种给药途径，对荷瘤小鼠进行治疗。结果显示以MSH-PE38KDEL1.1mg/只连续注射10天，可显著抑制肿瘤生长，其抑瘤率＞90%，且瘤周注射组肿瘤消失率为30%，静脉注射组肿瘤消失率为40%。病理切片HE染色结果显示MSH-PE38KDEL有效抑制了黑色素瘤B16细胞的肝内转移；电镜观察肝脏超微结构结果显示，MSH-PE38KDEL抑制了黑色素瘤B16细胞对机体细胞的破坏作用。重组毒素MSH-PE38KDEL在体内具有明显的抗黑色素瘤的效应，毒副作用低，病理及电镜结果显示对实质脏器均未见损伤。MSH-PE38KDEL有望成为靶向治疗黑色素瘤的候选药物。
Malignant melanoma is one of the malignant tumors with the fastest incidencegrowth. So far, the most effective therapy to the metastatic melanoma has not beendiscovered. Recombinant toxin is expected to become one of the most effective(assisting) therapy to melanoma for its high particularity and strong cytotoxity,especially to the metastatic melanoma. This study concentrated on the specificcombination of α-MSH with MC1R on the surface of malignant melanoma, as well asthe dozens of times’ gap between the binding site of MC1R on the surface ofmalignant melanoma and that of normal cells and other tumors, to make it as aspecific targeting carrier of malignant melanoma. The study also used pseudomonasexotoxin (PE) as the toxic part of immunotoxin, to build a recombinant toxin called asMSH-PE38KDEL, expressing melanoma targeting toxin in the way of geneticengineering.
Construction and expression of recombinant toxin—MSH-PE38KDEL. In this study,the linker of MSH and PE40was optimized by bioinformatics analysis, to ensure thattheir relative structure and function were not to be affected. Then PE40was modified,to reduce its immunogenicity (PE38) and increase its activity (PE38KDEL). Next thegene (MSH-PE38KDEL) was constructed and linked to the expression vectors—pET28a and pHis1525, to construct two recombinant plasmids—pET28a(+)-MSH-PE38KDEL and pHis1525-MSH-PE38KDEL, and they were expressed in theexpression host—Rosetta-gamiTM2(DE3) and WH320. By SDS-PAGE analysis andactivity determination, the recombinant plasmid with high expression andtoxicity—pET28a(+)-MSH-PE38KDEL was finally screened out. The plasmid wastranslated into Rosetta-gamiTM2(DE3) cell after it was determined with the correctsequence, and IPTG was used to induce fusion protein MSH-PE38KDEL expression,with SDS-PAGE as the determination on the expression products. Meanwhile, thesupporting software of gel image analysis system was applied to the analysis on expression level of MSH-PE38KDEL fusion protein, which took10%of the wholebacteria
Purification of recombinant toxin MSH-PE38KDEL. The Western Blot was usedto make somatic analyze after ultrasonic disruption to prove the soluble expression offusion protein. Purification methods such as somatic ultrasonic disruption,centrifugation, hydrophobic chromatography, ion exchange, molecular sievechromatography were adopted on the protein after expression. The purity quotient ofMSH-PE38KDEL with SDS-PAGE obtained over90%.
The in vitro antimelanoma activity of recombinant toxin MSH-PE38KDEL.The study tested the in vitro biological activity of human melanoma A875cells andmurine melanoma B16cells with high expression of α-MSH receptor, comparingthem with CNE, MCF, and2BS cells, whose α-MSH receptor expressions are low, inorder to prove its targeted killing effect. MTT test results showed that the killingeffect of MSH-PE38KDEL on melanoma cells A875and B16was over85%, and0onthe human normal2BS cells.
In vivo antimelanoma activity of recombinant toxin MSH-PE38KDEL. Themurine melanoma B16-cell was adopted in nude mice to establish a melanoma highlymetastatic animal model. The tumor-bearing mice were administrated with the routesof peri-tumor injection and intravenous injection. The result was that after10consecutive days of daily injection with the MSH-PE38KDEL concentration of1.1mg/mouse, the tumor growth was inhibited significantly with the rate of over90%.Meanwhile, the rate of disappearance in the group of peri-tumor injection was30%,and in the group of intravenous injection—that was40%. The HE staining resultshowed that MSH-PE38KDEL effectively inhibited the metastasis of melanoma B16cells in livers. The ultra structure of liver electron microscopy revealed thatMSH-PE38KDEL inhibited the damaging effects of melanoma B16cells on the livercells. Recombinant toxin MSH-PE38KDEL owned obvious antimelanoma effects invivo, with low toxic and side effects. The pathology results and electron microscopyrevealed no damage to the parenchymatous. In all, MSH-PE38KDEL is expected tobe adopted as one of the drug options to the targeting treatment for malignantmelanoma.
引文
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