摘要
目的:构建荷包猪SLA-2重链基因偶合底物肽(BSP)并研究其在pET-21a(+)中的蛋白表达。方法:设计荷包猪SLA-2-BSP复合基因引物,PCR扩增荷包猪SLA-2-HB-BSP复合基因,并克隆至pMD19-T Simple Vector,经酶切鉴定后,将SLA-2-HB-BSP复合基因与表达系统pET-21a(+)连接,并转化BL21(Rosetta)菌进行诱导表达,SDS-PAGE检测目的蛋白。结果:PCR结果显示,SLA-2-BSP大小为900bp左右,并成功克隆至pMD19-T Simple Vector载体,酶切后大小为876bp,该基因连接pET-21 a(+)并转化宿主菌BL21(Rosetta)后,经诱导表达和SDS-PAGE检测,目标蛋白大小为34.4kDa,经优化后目的蛋白相对表达含量达到了45%。结论:该研究成功构建荷包猪SLA-2偶合BSP的pET-21a重组表达系,为下一步构建猪SLAⅠ类分子四聚体奠定了基础。
Objective：To construct the heavy chain of SLA -2 conjugated with the Bio A suhstrate peptide （BSP）and to express the recom- binant genes in pET -21a（ ＋ ）for HeBao Pig. Method：A pair primer to express the recombinant SLA -2 -BSP was designed and the re- combinant SLA -2 -HB -BSP was amplified by PCR followed by sub -cloning the gene into pMD 19 -T Simple Vector. After identifi- cation by cleavage with Nde I and Xho I ,the SLA -2 - HB - BSP was ligated to pET -21a（ ＋ ）and the recombinant plasmids was trans- formed to BL21 （Rosseta） to be induced to express followed by analysis of the character of expressing products by SDS - PAGE. Reslflt ：The PCR results were shown that the length of nucleotides of SLA - 2 - HB - BSP was about 900 bp which was consistent with the calculated value. Then, the SLA -2 - HB - BSP with 876bp was successfully inserted into the pMD~ 19 -T Simple Vector identified by cleavage with EcoR V and Xho I and the constructed expressing vector was named as pET - 21a（ ＋ ）/SLA - 2 - HB - BSP. By SDS - PAGE, SLA - 2 - HB - BSP was successfully expressed in Escherichia coli BI21 （Rosseta） and the interest of protein was about 34. 4 kDa. After optimiza- tion, the relative expressing content of the interest of protein reached to 45%. Conclusion：Ii was concluded that the recombinant express- ing system containing SLA- 2 conjugated with BSP in pET- 21a（ ＋ ）was successfully constructed and the research will pave the base to construct tetramer in swine SLA class I .
出处
《生物技术》
CAS
CSCD
北大核心
2012年第5期17-20,共4页
Biotechnology
基金
国家自然基金项目("猪源病毒CTL多肽表位与SLA-I结晶研究"
No.31172304)
大连大学创新基金项目("猪SLA-2重链基因偶合底物肽及表达研究"
No.2011170)资助~~
