摘要
为获得大量的牛型分枝杆菌卡介苗热休克蛋白65(HSP65),研究了其发酵条件,分离纯化,并初步考察了其生物活性。应用PCR方法,克隆了HSP65基因,构建高效表达的pET-28a-HSP65重组质粒,转化大肠杆菌BL21,获得HSP65重组基因工程菌E.coliBL21/pET-28a-HSP65。工程菌经乳糖诱导4h后,重组蛋白HSP65得到高效表达,表达产物经硫酸铵沉淀、DEAE-Cellulose离子交换柱纯化;并初步考察了HSP65的免疫佐剂功能。工程菌在5mmol/L的乳糖诱导4h后,产物得到高效表达,经硫酸铵沉淀、DEAE-Cellulose离子交换纯化后可得到电泳纯的HSP65;初步的动物免疫发现,与HSP65偶联的VEGF121免疫原性有所提高。经发酵纯化后得到重组HSP65纯品,初步展现良好的免疫佐剂功能。
To obtain protein of HSP65 from BCG and to perform the purification as well as the activity determination, the whole gene of HSP65 was amplified by PCR and inserted into pEH28a and then transformed into E. coli BL21. After being induced with lactose for 4h, recombinant protein was expressed at the highest level. HSP65 was purified by precipitation with ammonium sulfate and DEAE-cellulose chromatography. Furthermore its function of immunoadjuvant was examined. The results showed that： 6h induction with lactose of 5mmol/L led to optimized expression of the recombinant protein. The product was purified by precipitation with ammonium sulfate and DEAE-Cellulose ion exchange chromatography. HSP65 was found to increase significantly the immunogenicity of VEGF121 conjugated with HSP65. Recombined HSP was expressed and purified successfully. It shows the modest capability of immunoadjuvant.
出处
《药物生物技术》
CAS
CSCD
2009年第1期19-23,共5页
Pharmaceutical Biotechnology
