Eimeria tenella表面抗原基因sag10和λMZP5-7在毕赤酵母中的表达
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摘要
鸡柔嫩艾美耳球虫(Eimeria tenella)主要寄生在宿主盲肠的粘膜下,引起出血性肠炎,致病力强,严重影响养殖业健康发展。目前使用抗球虫药是防治肉仔鸡病的主要手段,但抗药性和药物残留等问题限制了球虫药物的广泛应用。研究表明重组球虫抗原免疫鸡后能产生一定的保护力,保护性抗原基因是其免疫功效的决定因素。球虫表面抗原基因sag10、λMZP5-7是重要的候选基因之一。λMZP5-7的免疫保护作用已被证实,sag10基因在子孢子入侵和裂殖子生殖阶段均有表达,具有潜在的研究价值。本实验目的是在甲醇营养型毕赤酵母中表达Eimeria tenella表面抗原基因,为研制有效球虫基因工程疫苗提供新的途径。同时也为下一步进行免疫保护研究提供材料。
本文提取Eimeria tenella北京株第二代裂殖子总RNA,根据Genbank报道序列,编号L08257、AJ586552设计引物,应用RT-PCR技术扩增得到Eimeria tenella表面抗原基因λMZP5-7和sag10。与pGEM-T载体连接后测序,结果λMZP5-7与报道序列完全一致共948bp,编码315个氨基酸;pGEMT-sag10序列测定结果在NCBI上BLAST分析比较,结果表明,sag10基因编码序列与文献报道的序列同源性为99.01%,该球虫表面抗原基因全长708bp,编码236个氨基酸。其中有7个核苷酸与报道序列差异,4个氨基酸序列有差异。分别将λMZP5-7、sag10与真核表达载体pPIC9K连接,构建了pPIC9K-MZP和pPIC9K-sag10分泌型真核表达质粒,分别转化毕赤酵母GS115,利用G418抗性筛选多拷贝重组菌株,摇瓶水平进行诱导表达。SDS-PAGE和Western Blot进行检测,分别在大约36 KD和43kD处有明显的免疫印迹条带,结果MZP、SAG两种抗原蛋白得到表达,而且能被特异性抗体所识别,具有生物学活性。筛选到表达量最高的菌株S7在5L发酵罐进行优化表达,随着甲醇的诱导发酵上清中SAG10蛋白不断累积,当诱导120h后生物量达到最高354mg/mL,重组蛋白占总蛋白量的34%。
Cecal coccidiosis has tremendous impact on poultry throughout the world because of consequent mortality, morbidity and weight loss in affected birds. Control of Eimeria tenella in poultry is presently accomplished by prophylactic chemotherapy, but the rapid emergence of drug resistant parasites, coupled with difficulty and expense of developing new drugs, has led to a search for new approaches to coccidiosis control involving immunological, biotechnological and genetical methods. Of these, immunological approach is assuming more importance. The protective effect Eimeria tenella surface antigen geneλMZP5-7 has been confirmed and sag10 was expressed both in the stages of merozoites and sporozoites which were the most dangerous to the chicken.
The total RNA was extracted from the Beijing strain of Eimeria tenella and used as the template for the cloning of theλMZP5-7 and sag10 gene by RT-PCR with specific primers designed according to the reported sequence in Genbank( L08257、AJ586552). The expression plasmids pPIC9K-MZP and pPIC9K-sag10 of Pichia pastoris were constructed and transformed into Pichia pastoris GS115. Multi-copy Pichia pastoris strains were screened by G418. The selected strain was cultured in 100 ml flasks and the supernatant was analyzed by SDS-PAGE and Western Blot. The results indicated that the SAG and MZP5-7 protein were successfully expressed and secreted in a soluble form with a molecular weight of approximately 43 kDa and 36 kDa in recombinant Pichia pastoris , respectively ,when induced by 0.5%methanol. The selected strain S7 was ferment in 5L fermentor and the biomass was up to 354mg/mL when induced for 120h.,and the percentage of the recombinant protein was 34% in the tatol protein.
本文提取Eimeria tenella北京株第二代裂殖子总RNA,根据Genbank报道序列,编号L08257、AJ586552设计引物,应用RT-PCR技术扩增得到Eimeria tenella表面抗原基因λMZP5-7和sag10。与pGEM-T载体连接后测序,结果λMZP5-7与报道序列完全一致共948bp,编码315个氨基酸;pGEMT-sag10序列测定结果在NCBI上BLAST分析比较,结果表明,sag10基因编码序列与文献报道的序列同源性为99.01%,该球虫表面抗原基因全长708bp,编码236个氨基酸。其中有7个核苷酸与报道序列差异,4个氨基酸序列有差异。分别将λMZP5-7、sag10与真核表达载体pPIC9K连接,构建了pPIC9K-MZP和pPIC9K-sag10分泌型真核表达质粒,分别转化毕赤酵母GS115,利用G418抗性筛选多拷贝重组菌株,摇瓶水平进行诱导表达。SDS-PAGE和Western Blot进行检测,分别在大约36 KD和43kD处有明显的免疫印迹条带,结果MZP、SAG两种抗原蛋白得到表达,而且能被特异性抗体所识别,具有生物学活性。筛选到表达量最高的菌株S7在5L发酵罐进行优化表达,随着甲醇的诱导发酵上清中SAG10蛋白不断累积,当诱导120h后生物量达到最高354mg/mL,重组蛋白占总蛋白量的34%。
Cecal coccidiosis has tremendous impact on poultry throughout the world because of consequent mortality, morbidity and weight loss in affected birds. Control of Eimeria tenella in poultry is presently accomplished by prophylactic chemotherapy, but the rapid emergence of drug resistant parasites, coupled with difficulty and expense of developing new drugs, has led to a search for new approaches to coccidiosis control involving immunological, biotechnological and genetical methods. Of these, immunological approach is assuming more importance. The protective effect Eimeria tenella surface antigen geneλMZP5-7 has been confirmed and sag10 was expressed both in the stages of merozoites and sporozoites which were the most dangerous to the chicken.
The total RNA was extracted from the Beijing strain of Eimeria tenella and used as the template for the cloning of theλMZP5-7 and sag10 gene by RT-PCR with specific primers designed according to the reported sequence in Genbank( L08257、AJ586552). The expression plasmids pPIC9K-MZP and pPIC9K-sag10 of Pichia pastoris were constructed and transformed into Pichia pastoris GS115. Multi-copy Pichia pastoris strains were screened by G418. The selected strain was cultured in 100 ml flasks and the supernatant was analyzed by SDS-PAGE and Western Blot. The results indicated that the SAG and MZP5-7 protein were successfully expressed and secreted in a soluble form with a molecular weight of approximately 43 kDa and 36 kDa in recombinant Pichia pastoris , respectively ,when induced by 0.5%methanol. The selected strain S7 was ferment in 5L fermentor and the biomass was up to 354mg/mL when induced for 120h.,and the percentage of the recombinant protein was 34% in the tatol protein.
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