cDNA芯片结合抑制性差减杂交技术构建对虾白斑综合征病毒表达基因和螯虾免疫相关基因文库

Construction the cDNA library of expression profiles of penaeid shrimp WSSV and immune genes of crayfish with suppression subtractive hybridization and cDNA chip

以克氏原螯虾做为对虾白斑综合征病毒(WSSV)的增殖模型,试验组接种WSSV,对照组接种PBS。分别以试验组为检测组(tester)、对照组为驱逐组(driver),进行正向抑制性差减杂交;以对照组为tester、试验组为driver,进行反向SSH。将获得的正、反向差减杂交产物克隆入质粒载体PinPointTM Xa-1T-Vector,再转化大肠杆菌DH5α,共构建了2个分别含1620和400个克隆的正、反向差减文库。经PCR扩增,1514个差减克隆得到产物,其中正向差减克隆1221个,反向差减克隆293个。获得的PCR产物经浓缩、变性处理,用自动点样仪点制于尼龙膜载体上,制成cDNA芯片,用cDNA芯片对SSH获得的差减克隆进行鉴定,共获得差异表达克隆278个,其中攻毒组高表达克隆255个;对照组高表达克隆23个。

The cDNA of crayfish （ Procambarus clarkii ） infected with white spot syndrome virus（WSSV） were used as tester and that of the control as driver to construct a forward subtraction. A reverse subtraction was performed by used tester as driver and driver as tester at the same time. The subtracted products were cloned into the PinPointTM Xa-I T-Vector. Then the recombinated plasmid was transformated into DH5α. A forward subtracted cDNA library including 1620 clone and a reverse subtracted cDNA library including 400 clone were constructed respectively. Using sequences flanking the cloning site as primers, 1514 clones were amplified from the 2020 clones constructed by suppression subtractive hybridization. 1221 clones of these were got from forward subtraction library and 293 clones from reverse subtraction library. After concentration and degeneration the PCR fragments were robotically printed onto nylon membrane to make a cDNA chip. In the end, 255 positive clones from forward subtraction library and 23 positive clones from reverse subtraction library were detected by cDNA chip.