摘要
人白介素-11(Interleukin-11,IL-11)在体内由PU-34细胞产生,具丝裂原活性、能支持IL-6依赖的浆细胞瘤细胞系T1165生长。最基本的功能在于造血调控作用,在体外对不同阶段的巨核细胞和血小板生成都具有特异的促进作用。体内注射hIL-11可显著刺激巨核细胞集落(CFU-MK)生长和增加血小板计数,还具有促进消化道上皮损伤恢复、调节脂肪细胞分化等多种生物活性。但是天然的白介素-11在在体内的半衰期较短,为了达到延长其在体内的半衰期,本研究采用了把白介素-11和人血清白蛋白以无连接肽的方式融合。
取人胎肺成纤维细胞,以DMEM刺激培养,提取总RNA,通过反转录得到编码人白介素-11的cDNA,克隆到T载体pMD19-Simple上,构建含有IL-11编码基因的重组质粒pMD19/IL-11,DNA序列测定结果证明插入序列与IL-11编码序列完全一致。
分别以pMD19/IL-11和含有编码HSA编码基因的重组质粒pBlue/HSA为模板,利用重叠PCR的办法,扩增得到HSA-IL-11融合基因,连接到载体上得到重组质粒pBlue/HI;对pBlue/HI和穿梭质粒pPIC9K分别用EcoRⅠ/NotⅠ进行双酶切,将HSA-IL-11融合基因克隆到表达质粒pPIC9K中构建重组表达质粒pPHI11;经测序验证,所得到的表达载体中目的片段碱基序列和预期一致,读框正确。
将SalⅠ线性化的pPHI11电转化巴斯德毕赤酵母(Pichia pastoris)GS115,通过组氨酸缺陷型标记和G418抗性筛选,得到抗0.75 mg/mL的G418的毕赤酵母转化子,经过PCR验证,转化子中含有HSA-IL-11融合基因。
BMGY种子培养基培养细胞,经离心收集后,于BMMY诱导表达培养基在30℃,200 r/min条件下,以甲醇为唯一碳源诱导3 d,经聚丙烯酰胺凝胶电泳(SDS-PAGE)检测,重组子表达出相应分子量的目标蛋白,HSA及IL-11抗体的Western-blot免疫杂交实验检测结果表明,融合蛋白中含有HSA和IL-11,说明HSA-IL-11融合蛋白得以表达。定量测定结果表明,融合蛋白在诱导上清液中的表达量约为120 mg/L。
对重组菌在摇瓶转速、诱导甲醇浓度、诱导时间、诱导pH、诱导温度等条件下进行优化,得到的诱导条件为:诱导温度30℃,甲醇浓度5%,pH6,诱导细胞密度O.D.600=60,在上述优化条件下HSA-IL-11的表达量为198 mg/L。
B9-11/MTT法检测诱导上清液生物学活性的结果表明,融合蛋白HSA-IL-11可以有效刺激B9-11细胞增殖,其IL-11生物学活性约为6.2×104 U/mg。
Human interleukin-11(IL-11) is produced by PU-34 cells. It was initially described as a growth factor synergising with other factors in the regulation of hematopoiesis. The half-life of native IL-11 is short. In order to prolong the half-life, the fusion protein HSA-IL-11 was constructed by splicing the IL-11 to Human Serum Album (HSA) without peptide linker.
The cDNA encoding IL-11 was obtained by RT-PCR with the template of total RNA purified from human fetal lung fibroblast, which was prepared from fresh tissue. These cells were stimulated with DMEM, then cloned into pMD19-Simple to construct the recombinant plasmid pMD19/IL-11.
The fusion gene encoding HSA-IL-11 was amplified by overlap PCR extension using pMD19/IL-11 and pBlue/HSA (containing the gene encoding HSA) as the template, and then cloned into pBluescriptⅡKS(+) to construct the recombinant plasmid pBlue/HI. The pBlue/HI was digested with EcoRI and NotI, and ligated between the corresponding sites of pPIC9K, yielding the recombinant expressing plasmid pPHI11. Fidelity of the cloned DNA fragment was confirmed by DNA sequencing.
The linearized plasmid pPHI11 digested by SalI was transformed into Pichia pastoris GS115 by electroporation. The transformants resisting 0.75 mg/mL of G418 were obtained. It was confirmed by PCR amplification that there was fusion gene encoding HSA-IL-11 in the transformant of P. pastoris GS115(pPHI11).
The recombinant yeast of P. pastoris GS115(pPHI11) cultivated in BMGY medium and collected by centrifugation was resuspended in expression medium of BMMY, induced with methanol as the sole carbon source for 3 days at 30℃. SDS-PAGE analysis showed that the recombinant yeast expressed the protein with the similar molecular weight to the target fusion protein. Western-blot result was indicated that the expressed fusion protein HSA-IL-11 contained antigen both IL-11 and HSA. Quantitative analysis showed that the expression level of HSA-IL-11 was about 120 mg/L in expression suspension.
The result of orthogonal test for rotation speed, temperature, pH, methanol concentration, and time showed the optimum expression condition for the recombinant yeast was as follows: inducing temperature of 30℃, methanol concentration of 5%; pH6. The expressing level was increased to 198 mg/L.
The bioactivity of the expression supernatant was checked by B9-11/MTT method and the result showed that the fusion protein HSA-IL-11 can stimulate the division of B9-11 cells. The IL-11 activity was about 6.2×104 U/mg of the supernatant.
引文
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