猪乳铁蛋白基因克隆及其在P.methanolica系统中的表达研究  

Cloning of recombinant porcine lactoferrin and its expression in a P.methanolica system

作　　者：刘光富[1] 刘明启[1] 

机构地区：[1]中国计量学院生命科学学院,杭州310018

出　　处：《黑龙江畜牧兽医》2012年第10期10-13,17,162,共6页Heilongjiang Animal Science And veterinary Medicine

基　　金：浙江省教育厅科研计划项目(Y201018892)

摘　　要：为了研究猪乳铁蛋白(PLF)基因克隆及其在酵母(P.methanolica)系统中的表达,试验从泌乳15 d的约克夏母猪乳腺组织中提取总RNA,应用RT-PCR技术扩增猪乳铁蛋白基因,获得1条长为2 115 bp的目的片段,将目的基因克隆到酵母表达载体pMET-B中,采用电转化法转化P.methanoli-ca感受态细胞PMAD11;选取Ade+MutS重组子进行诱导培养,产物纯化后进行SDS-PAGE和West-ern-blot分析及其他生物学特性分析。结果表明:rPLF在P.methanolica表达系统中表达,表达产物的分子质量约为78.0 ku;在第144小时时表达量最高,最佳pH值为7.0,最佳铁离子浓度为100μmol/L;重组PLF对大肠杆菌ATCC25922有抑菌效果,而且抑菌效果随着浓度的升高随之增强。To study the cloning of recombinant porcine lactoferrin （PLF） and its expression in a Pichia methanolica system, a total RNA from mammary gland cells of 15 - day - lactating Yorkshire sow was extracted. The PLF gene was cloned by the reverse transcription - coupled polymerase chain reaction （ RT - PCR）, and then a DNA fragment about 2 115 bp in length was obtained, and the PCR product was cloned into P. methanolica expression vector pMET - B. The recombinant plasmid pMET - B - PLF was transformed into PMAD11 competent cells by electro- potation, and Ade ＋ （ Muts ） recombinants were induced with methanol, then the recombinant protein was purified and analyzed by SDS - PAGE and Western - blot. The results showed that the recombinant porcine lactoferrin （rPLF） was well expressed in P. methanolica, and its molecular weight was estimated to be approximately 78.0 ku. The expression amount of rPLF in the culture medium reached to the maximum at 144 hours, the optimum pH was 7.0 , and the optimum concentration of iron ions was 100 ppm. The results indicate that the rPLF has a bacterlostatic effect on the E. coli ATCC25922, which increases with the increase of the concentration.

关 键 词：猪乳铁蛋白 酵母 克隆 表达 

分 类 号：Q344.13[生物学—遗传学]