人磷酸二酯酶同工酶3A在昆虫杆状病毒系统中的表达

Expression of human phosphodiesterase 3A gene using baculovirus expression system in insect cell

目的利用杆状病毒表达系统探讨人磷酸二酯酶同工酶3A(HPDE3A)在Tn细胞中的表达。方法用重组HPDE3A杆状病毒感染昆虫Tn细胞进行蛋白表达,通过RTPCR,SDSPAGE和Westernblotting技术检测HPDE3A表达情况。结果重组杆状病毒在感染Tn细胞后形态变化明显,获得表达产物,RTPCR扩增结果证实了Tn细胞中HPDE3A的mRNA水平增高,Tn细胞能够表达出与HPDE3A多克隆抗体结合的蛋白,蛋白分子质量约为120kD。结论HPDE3A在昆虫杆状病毒表达系统中可以稳定表达,为进一步研究HPDE3A的生物学活性及筛选HPDE3A抑制剂奠定了基础。

Aim To investigate the expression of recombinant human phosphodiesterase 3A （HPDE3A） using baculovirus expression system in Tn cell line. Methods The HPDE3A cDNA was recombined with baculovirus, and then the recombinant was transfected into Tn cell line. The expression of HPDE3A in Tn cell line was detected and identified by the RT-PCR, SDS-PAGE and Western blotting. Results The recombinant HPDE3A protein was stably expressed in Tn cell line and detected by the distinct morphological changes of Tn cell, RT-PCR, SDS-PAGE and Western blotting using polyclonal antibody. The Mw of the recombinant protein was about 120 kD. Conclusion Recombinant HPDE3A can be expressed in Tn cell line using the baculovirus expression system, and thus provided the basic material for studying its bioactivity and application in screening for HPDE3A inhibitor.