Recognition of signal peptide by protein translocation machinery in middle silk gland of silkworm Bombyx mori

To investigate the functions of signal peptide in protein secretion in the middle silk gland of silkworm Bombyx mori, a series of recombinant Autographa californica multiple nucleopolyhedroviruses containing enhanced green fluorescent protein （egfp） gene, led by sericin-1 promoter and mutated signal peptide coding sequences, were constructed by region-deletions or single amino acid residue deletions. The recombinant Autographa californica multiple nucleopolyhedroviruses were injected into the hemocoele of newly ecdysed fifth-instar silkworm larvae. The expression and secretion of EGFP in the middle silk gland were examined by fluorescence microscopy and Western blot analysis. Results showed that even with a large part （up to 14 amino acid residues） of the ser-1 signal peptide deleted, the expressed EGFP could still be secreted into the cavity of the silk gland. Western blot analysis showed that shortening of the signal peptide from the C-terminal suppressed the maturation of pro-EGFP to EGFP. When 8 amino acid residues were deleted from the C-terminal of the signal peptide （mutant 13 aa）, the secretion of EGFP was incomplete, implicating the importance of proper coupling of the h-region and c-region. The deletion of amino acid residue（s） in the h-region did not affect the secretion of EGFP, indicating that the recognition of signal peptide by translocation machinery was mainly by a structural domain, but not by special amino acid residue（s）. Furthermore, the deletion ofArg^2 or replacement with Asp in the n-region of the signal peptide did not influence secretion of EGFP, suggesting that a positive charge is not crucial.     

苜蓿丫纹夜蛾核型多角体病毒fp25k基因的改造及用于稳定转化Sf9昆虫细胞系

【目的】苜蓿丫纹夜蛾核型多角体病毒(Autographa californica multicapsid nucleopolyhedrovirus, AcMNPV)在昆虫细胞中连续传代以后,会出现从多多角体表型到少多角体表型的转变,这种转变与一个编码25 kDa蛋白的基因（few polyhedra, fp25k）突变失活有关。杆状病毒的fp25k基因突变后产生的包涵体（多角体）衍生病毒粒子变少而出芽型病毒粒子增加,会降低外源基因在杆状病毒表达系统中的表达。本研究拟改造fp25k并构建能持续表达FP25K蛋白的转基因昆虫细胞,以克服杆状病毒, fp25k基因易突变导致的表达系统缺陷。【方法】本实验通过改造杆状病毒, fp25k基因在细胞传代过程中容易产生突变的位点,得到 mfp25k,并将mfp25k构建到pIZT/V5-His载体上,重组载体转染Sf9细胞,通过Zeocin抗性筛选逐步淘汰未成功转化的Sf9细胞。【结果】成功改造AcMNPV的, fp25k基因的TTAA位点,得到pIZT-mfp25k重组载体。重组载体成功转染Sf9细胞,通过Zeocin抗性筛选后获得基因组中带有mfp25k的Sf9-mfp25k稳定的转基因细胞系。用AcMNPV的fp25k突变型病毒AcP2感染转基因Sf9-mfp25k昆虫细胞系与正常Sf9细胞,发现转基因Sf9-mfp25k昆虫细胞系表达的FP25K蛋白可弥补病毒, fp25k基因突变的缺陷。【结论】建立的Sf9-mfp25k转基因昆虫细胞系通过细胞表达FP25K蛋白,可以弥补因杆状病毒fp25k基因突变产生的缺陷。研究结果为构建稳定的杆状病毒 昆虫细胞表达系统提供了新途径。     

A defective viral genome maintained in Escherichia coli for the generation of baculovirus expression vectors

A novel baculovirus genome has been generated that can be maintained in Escherichia coli and which facilitates the rapid and efficient generation of recombinant baculovirus expression vectors. This genome, designated bAcGOZA, lacks a portion of the essential ORF1629 gene but includes a miniF replicon and selectable kanamycin-resistance marker. The bAcGOZA can replicate only in E. coli and can be rescued by recombination with a transfer vector containing an intact ORF1629. There is no background of non-recombinant virus.     

Construction and Characterization of a Novel Bacmid AcBac-Syn Based on a Synthesized Baculovirus Genome

Virologica Sinica - Baculoviruses are large DNA viruses which have been widely used as expression vectors and biological insecticides. Homologous recombination and Bac-to-Bac system have been the...