中国家蚕抗菌肽CBM1、CBM2的克隆和表达研究

根据所测定的中国产家蚕CBM1和CBM2 cDNA序列，设计成熟肽的特异性引物，将此基因与凝血因子Xa（FXa）的切割序列以PCR方法连接起来，克隆至pGEX—KG表达质粒中。该载体为改进的GST融合表达质粒，所携带的6个Gly有助于提高FXa的切割效率。克隆得到的融合载体在大肠杆菌JM109中扩增和筛选。经DNA测序，证实为含有正确序列的乙酰化的pGEX—KG—FXa—NCBM1（pKG—FXa—NCBM1）、非乙酰化的pGEX—KG—FXa—CBM1（pKG—FXa—CBM1）和乙酰化的pGEX—KG—FXa—NCBM2（pKG—FXa—NCBM2）、非乙酰化的pGEX—KG—FXa—CBM2（pKG—FXa—CBM2）。将该载体在BL21中诱导表达，得到GST-FXa切割位点NCBM1、CBM1、NCBM2、CBM2等4种融合蛋白。经SDS—PAGE，考马斯亮兰染色后，Lab—Work扫描，其表达产物达到15％～20％，每升培养液平均可得20mg融合蛋白。菌体总蛋白经GST-亲和层析柱得到纯的GST—FXa—NCBM1、GST—FXa—CBM1、GST—FXa—NCBM2、GST—FXa—CBM2融合蛋白，再经FXa酶切后，透析过柱得到重组的NCBM1和CBM1。平板抑菌试验证明，4种融合蛋白均有明显的生物学活性，且没有显著的差异。

Expression and Study of Amidating Antibacterial Peptides and No Amidating Antibacterial Peptides in the Silkworm from Chinese

According to cDNA sequence of cecropin CBM1 and CBM2, specific primers of mature peptides of CBM1 and CBM2 were designed. CBM1 and CBM2 genes were cloned into pGEX-KG which contains the sequence PGISGGGGG whose linker greatly increases FXa cleavage efficiency, and these plasmids are transformed into E.coli JM109 and position clones were obtained by PCR and sequencing. Four expression vectors, pGEX-KG-FXa-NCBM1 with an Asn codon, pGEX-KG-FXa-CBM1, pGEX-KG-FXa-NCBM2 with an Asn codon and pGEX-KG-FXa-CBM2 were obtained. Fusion proteins of the four kinds of plasmid were induced with IPTG in E.coli BL21. After FXa cleavage, CBM1, NCBM1, CBM2 and NCBM2 were purified. They all have antibacterial activity with inhibition zone assay of plate and they have no obvious difference each other. The results proved amidating in the C-terminus may be not necessary.