中国家蚕抗菌肽人工合成类CMⅣ基因在甜菜夜蛾虫体中的表达

EXPRESSION OF THE ARTIFICIAL CECROPIN CMⅣ-LIKE GENE OF CHINESE SILKWORM BOMBYX MORI IN LAPHYGMA EXIGUA LARVAE

将人工合成的中国家蚕抗菌肽类CMⅣ基因与抗菌肽信号肽基因连接 ,经EcoRⅠ、HindⅢ双酶切后 ,克隆于pFASTBacⅠ的EcoRⅠ、HindⅢ酶切位点之间 ,得到重组转座载体pFASTBac ABP ,经测序证明阳性克隆正确。将重组转座载体转化HD1 0Bac大肠杆菌 ,得到重组Bacmid ABP。将重组Bacmid转染sf2 1细胞及感染甜菜夜蛾 (Laphygmaexigua)幼虫 ,在培养细胞上清及虫体血淋巴中均测到抗菌活性。经Northernblotting证明感染甜菜夜蛾幼虫中有类CMⅣmRNA的存在。且表达产物在酸性电泳中电泳行为与天然抗菌肽CMⅣ组分相似。为进一步利用昆虫细胞及虫体生产抗菌肽药物打下了基础。

The synthesized CMⅣ-like Gene was linked with the signal peptide gene of nature silkworm antibacterial peptide and was inserted into baculovirus expression vector pFastBac 1,constructing a recombinant transposing vector.The vector was transformed into DH10Bac competent E.coli cells.The recombinant Bacmid was obtained.The recombinant Bacmid was transfected into sf21 cells to get the recombinant virus.Laphygma exigua larvae were infected with the recombinant virus to express the antibacterial peptide.The hemolymph were tested to have antibacterial activity.The active antibacterial peptide was purified by acid polyacrylamide electrophoresis.The specific expression of mRNA of CMⅣ-like Gene was tested using Northern blotting.