戊型肝炎病毒开放读框2的克隆及表达载体的构建

戊型肝炎病毒（HEV）为单股正链RNA病毒,整个基因组有3个开放性阅读框架（ORF）,ORF2（全长约1.98kb）是主要结构基因编码区。将经过PCR获得的ORF2基因插入非分泌型酵母穿梭质粒pPIC3,构成受醇氧化酶（AOX2）的启动子与转录终止区控制的酵母表达质粒,重组质粒经酶切线性化后转化酵母细胞GS115,经过鉴定可挑取与酵母染色体发生交换的重组体,用于进一步的真核表达。     

Characterization of the ksgA gene of Escherichia coli determining kasugamycin sensitivity

In the plasmid pUC8ksgA7, the coding region of the ksgA gene is preceded by the lac promoter (Plac) and a small open reading frame (ORF). This ORF of 15 codons is composed of nucleotides derived from the lacZ gene, a multiple cloning site and the ksgA gene itself. The reading frame begins with the ATG initiation codon of lacZ and ends a few nucleotides beyond the ATG start codon of ksgA. The ksgA gene is not preceded by a Shine-Dalgarno (SD) signal. Cells transformed with pUC8ksgA7 produce active methylase, the product of the ksgA gene. Introduction of an in-phase TAA stop codon in the small ORF abolishes methylase production in transformed cells. On the plasmid pUC8ksgA5, which contains the entire ksgA region, the promoter of the ksgA gene was found to reside in a 380 base pair Bgl1-Pvu2 restriction fragment, partly overlapping the ksgA gene, by two independent methods. Cloning of this fragment in front of the galK gene in plasmid pKO1 stimulates galactokinase activity in transformants and its insertion into the expression vector pKL203 makes beta-galactosidase synthesis independent of the presence of Plac. The sequence of the Bgl1-Pvu2 fragment was determined and a putative promoter sequence identified. An SD signal could not be distinguished at a proper distance upstream from the ksgA start codon. Instead, an ORF of 13 codons starting with ATG in tandem with an SD signal and ending 4 codons ahead of the ksgA gene was identified. This suggests that translation of the ORF is required for expression of the ksgA gene.(ABSTRACT TRUNCATED AT 250 WORDS)