摘要
本文报道建立以胸腺嘧啶核苷激酶(TK)为标记,利用痘苗病毒 TK 基因为旁侧序列的外源基因载体,通过体内同源重组的原理将外源基因导入臭曲霉菌,并用5-溴脱氧尿嘧啶核苷(BUdR)进行转化株选择的新系统,对黑曲霉菌 TK^+株的挑选、转化及选择条件等进行了研究。用这一系统,成功地将臭曲霉菌自身分离到的启动基因 H8驱动下的乙肝表面抗原(HBsAg)基因导入臭曲霉菌。Southern blot 和多次孢子传代证实,HBsAg 基因已与宿主的 DNA 稳定整合;在发酵上清液中,ELIAS 检测 HBsAg 为阳性(P/N 值在20左右),Western blot 有特异的带存在,免疫电镜观察发现,在发酵液中存在有与人血清中 HBsAg 颗粒大小、形态相似的 22nm 的颗粒。这些结果表明,HB(?)Ag 基因在臭曲霉菌中已获表达。本文建立的基因导入和选择系统可广泛应用于各种外源基因在臭曲霉菌中表达的研究。
A new system for selection of transfor-med Aspergillus foetidus was reported.Inthis system,TK^- A.foetidus which were con-structed by homologous recombination of mu-tated TK gene of vaccinia virus with TKgene of A.foetidus were screened by addingBUdR in agar plates.Conditions for screenof TK^+ A.foetidus strain,transformation ofA.foetidus and selection of transformed TK^-A.foetidus have been studied.By using thissystem,several transformed A.foetidus whichcontained HBsAg gene drived bf a promoterH8 cloned from genomic DNA of A.foeti-dus were isolated.It was demonstrated thatHBsAg gene was integrated into the chromo-some DNA of A.foetidus by Southern blotafter many passages of spores.ELISA show-ed that HBsAg was positive in the growthmedium (p/n=20).The 22nm particleswhich were very similar to the HBsAg parti-cles in human serum were found in thegrowth medium by immunoelectromicroscope.Western blot also gave the specific bands.Allthese data showed that HBsAg gene was ex-pressed in A.foetidus and the products weresecreated into the growth medium.The selec-tion system using TK gene as marker couldgenerally be used to study the expression offoreign gene in A.foetidus.
出处
《微生物学报》
CAS
CSCD
北大核心
1990年第2期98-104,共7页
Acta Microbiologica Sinica
基金
863”资助项目
关键词
臭曲霉
外源基因
TK基因
选择系统
Aspergillus foetidus
Thymidine Kinase
Promoter H8
HBsAg
