C型产气荚膜梭菌α毒素基因的克隆与表达

利用PCR技术,从C型产气荚膜梭菌染色体基因组中扩增了1．2kb的α毒素基因,将纯化的PCR产物与载体pGEM-T连接,转化至受体菌JM109中,经NcoI／EcoRI和BamHI／EcoRI酶切鉴定及核苷酸序列测定证实,重组质粒pXCPAl中含有α毒素全基因。随后用NcoI／EcoRI酶切质粒pXCPAl,回收α毒素基因片段,插入到事先经同样酶切处理的载体pET-28c中相应酶切位点,构建了表达质粒pETXAl,经NcoI／EcoRI和BamHI／EcoRI酶切鉴定及核苷酸序列测定证实,表达质粒含有α毒素基因且基因序列和阅读框架正确。重组菌株BL21(DE3)(pETXAl)表达产物经ELISA检测和SDS-PAGE分析,重组菌株表达的α毒素蛋白能够被α毒素单抗识别,其表达量占菌体总蛋白相对含量的16．28％。     

Development and characterization of a xylose-inducible gene expression system for Clostridium perfringens

A xylose-inducible gene expression vector for Clostridium perfringens was developed. Plasmid pXCH contains a chromosomal region from Clostridium difficile (xylR-P(xy)(lB)): xylR, encoding the xylose repressor, xylO, the xyl operator sequence, and P(xylB), the divergent promoter upstream of xylBA encoding xylulo kinase and xylose isomerase. pXCH allows tightly regulated expression of the chloramphenicol acetyltransferase reporter and the α-toxin genes in response to the inducer concentration. Thus, pXCH could constitute a new valuable genetic tool for study of C. perfringens.     

Cloning and sequencing of a phospholipase C gene of Clostridium perfringens

The gene encoding phospholipase C (alpha-toxin) of Clostridium perfringens was cloned into lambda gt10. The maximal size of the coding region was 1.4 kb and the minimum was 1.1 kb as determined by subcloning into the vector pBR322 and testing for activity. The nucleotide sequence of this region contained a single open reading frame of 1194 bp corresponding to a protein of Mr 45473 with a possible N-terminal signal sequence of 28 amino acids which when removed, would give a mature protein of Mr 42521. This is in good agreement with the reported size of 43 kDa. The coding region has a dG + dC content of 33.7%, and the codon usage displays a pronounced preference for codons with the lowest dG + dC content.     

Characterization of a transcriptional promoter of human papillomavirus 18 and modulation of its expression by simian virus 40 and adenovirus early antigens

RNA present in cells derived from cervical carcinoma that contained human papillomavirus 18 genomes was initiated in the 1.053-kilobase BamHI fragment that covered the complete noncoding region of this virus. When cloned upstream of the chloramphenicol acetyltransferase gene, this viral fragment directed the expression of the bacterial enzyme only in the sense orientation. Initiation sites were mapped around the ATG of open reading frame E6. This promoter was active in some human and simian cell lines, and its expression was modulated positively by simian virus 40 large T antigen and negatively by adenovirus type 5 E1a antigen.     

Negative and positive regulation in trans of gene expression from adeno-associated virus vectors in mammalian cells by a viral rep gene product.

We previously described use of the human parvovirus, adeno-associated virus (AAV), as a vector for transient expression in mammalian cells of the gene for chloramphenicol acetyltransferase (CAT). In the AAV vector, pTS1, the CAT gene is expressed under the control of the major AAV promoter p40. This promoter is embedded within the carboxyl-terminal region of an open reading frame (orf-1) which codes for a protein (rep) required for AAV DNA replication. We show here that the rep product has additional trans-acting properties to regulate gene expression. First, deletion or frame-shift mutations in orf-1, which occurred far upstream of p40, increased expression of CAT in human 293 (adenovirus-transformed) cells. This increased CAT expression was abolished when such mutant AAV vectors were transfected into 293 cells together with a second AAV vector which could supply the wild-type AAV rep product in trans. Thus, an AAV rep gene product was a negative regulator, in trans, of expression of CAT in uninfected 293 cells. In adenovirus-infected 293 cells, the function of the AAV rep product was more complex, but in some cases, it appeared to be a trans activator of the expression from p40. In HeLa cells, only trans activation by rep was seen in the absence or presence of adenovirus. Neither activation nor repression by the rep product required replication per se of the AAV vector DNA. Thus, trans-acting negative or positive regulation of gene expression by the AAV rep gene is modulated by factors in the host cell and by the helper adenovirus.     

A型产气荚膜梭菌α毒素基因表达及其免疫保护作用的初步研究

利用PCR技术,从A型产气荚膜梭菌标准株染色体DNA中扩增出α毒素基因,构建了含α毒素基因的重组菌株BL21(DE3)(pXETA02)。经酶切鉴定和序列测定证实,构建的表达质粒pXETA02含有α毒素基因序列。经SDS-PAGE、Western blot分析和ELISA检测,重组菌株表达的α毒素蛋白能够被α毒素单抗识别。表达优化结果表明,以IPTG为诱导剂诱导α毒素表达的优化条件是:培养基pH 7.5,培养温度37℃,IPTG浓度0.8mmol/L,菌体生长密度OD600达到0.8时加入IPTG,诱导时间5h,此时α毒素蛋白表达量为34.83%。以乳糖为诱导剂诱导α毒素表达的优化条件是:培养基pH7.5、培养温度37℃,乳糖浓度0.1g/L,菌体生长密度OD600达到0.8时加入乳糖,诱导时间5h,α毒素蛋白表达量为23.82%。动物实验结果表明,用重组菌株α毒素蛋白免疫的小鼠可以抵抗1MLD的A型产气荚膜梭菌标准株C57-1毒素攻击。