摘要
目的将恶性疟原虫裂殖子表面蛋白1C末端msp1-42基因(3D7株)导入烟草叶绿体基因组中并进行同质化筛选,为利用叶绿体表达系统生产MSP1-42蛋白提供基础材料。方法利用烟草偏爱密码子设计克隆恶性疟原虫(3D7株)msp1-42基因的引物,从含msp1-42基因的pBluntmsp质粒中扩增出msp1-42,构建烟草叶绿体表达载体LRrrmsp。通过基因枪转化法转化烟草叶片,在500mg/L壮观霉素的选择压力下筛选抗性植株,采用PCR鉴定抗性植株的msp1-42基因及aadA基因,对鉴定阳性植株进行同质化筛选(叶片切碎、在含500mg/L壮观霉素分化培养基上分化出新的植株)3轮以上,并采用多重PCR分析其同质化情况。结果构建了恶性疟原虫msp1-42基因的叶绿体表达载体LRrrmsp。基因枪转化后获得6个转化子,转化频率为0.6个/枪。转化3～5d后,小块叶片开始增大增厚,并逐渐由绿色变为黄绿色,7～10d后黄化或白化,再经约30d的筛选培养,叶片上出现绿色小芽。PCR检测抗性植株的msp1-42及aadA基因,分别扩增出约900bp与500bp的条带,与预期相符。多重PCR分析从经过3轮同质化筛选植株的叶绿体基因中扩增出与未转基因烟草对照大小一致的弱条带,表明经过3轮同质化筛选的转基因植株仍含有未插入外源基因的叶绿体基因组。结论获得含恶性疟原虫msp1-42的烟草叶绿体表达载体,并将恶性疟原虫msp1-42基因导入烟草叶绿体基因组中,获得尚未完全同质化的转基因烟草。
Objective To construct chloroplast expression vector, and introduce the C-terminal region of the merozoite surface protein 1 gene （msp 1-42） of Plasmodium falciparum 3D7 strain into the chloroplast genome of tobacco for expression of the recombinant protein MSP 1-42. Methods Forward and reverse primers, adjusted to tobacco chloroplast codon preferences, were used for generation of msp1-42 gene from pBluntmsp plasmid which contains msp1- 42 gene. A chloroplast expression vector LRrrmsp was constructed and bombarded into leaves of tobacco by a biolistic He particle delivery system. Media containing 500 mg/L spectinomycin were used for selection of spectlnomycin resistant plant. PCR was carried out to check the introduction of the msp1-42 and aadA genes into the chloroplast genome. The transgenic plants with rasp 1-42 and aadA gene insertion were cut and cultured on the generation MS media containing 500 mg/L spectinomycin for at least 3 turns, and multiple PCR were applied to analyse their homogenization. Results A chloroplast expression vector LRrrmsp was constructed and confirmed with PCR and enzyme digestion analysis. Six transformmants were obtained with a transformation rate 0.6/gun. The rasp 1-42 and aadA genes were amplified from spectinomycin resistant plants by PCR detection. Wild type chloroplast gene was detected by multiple-PCR analysis. Conclusion A chloroplast expression vector containing msp1-42 gene was constructed. The mspl-42 gene was introduced into chloroplast genome of tobacco and heterogenous transgenic tobacco was obtained.
出处
《中国寄生虫学与寄生虫病杂志》
CAS
CSCD
北大核心
2008年第4期263-267,共5页
Chinese Journal of Parasitology and Parasitic Diseases
关键词
恶性疟原虫
叶绿体
基因表达
裂殖子表面蛋白1
植物疫苗
Plasmodium falciparum
Chloroplast
Gone expression
Merozoite surface protein 1
Plant vaccine
