鸡柔嫩艾美耳球虫体外模型的建立及其HSP70基因的克隆表达
摘要
鸡球虫病是由艾美耳属的柔嫩艾美耳球虫(Eimeria tenella)等7种球虫单独或混合感染肠道不同部位所引起的一种肠道细胞内寄生性原虫病。
(1)本研究利用鸡胚肠上皮细胞来替代鸡肾细胞,建立一种新型的鸡球虫体外细胞培养模型,因为球虫感染肠上皮细胞的条件和过程更加接近了鸡体自然感染状态。探讨了鸡胚肠上皮细胞体外分离培养的方法,结果显示,鸡胚肠上皮细胞,在37℃用Ⅰ型胶原酶分步消化70 min,低速离心后接种培养板,90 min后将未贴壁的细胞移至新的有胶原铺底的24孔培养板中,5%CO2 39.5℃,DMEM含5%胎牛血清的条件下培养,24～48 h细胞贴壁,7～8 d长成单层。
(2)收集纯化的球虫子孢子,接种于适合球虫生长发育的鸡胚肠上皮原代细胞,采用这一细胞模型探索了E.tenella裂殖生殖阶段的发育情况,经试验确定了最适球虫繁殖的培养基和添加成分,建立了艾美耳球虫鸡胚肠上皮细胞培养适应株。结果表明,在细胞培养第8天时接种子孢子,子孢子在接种2 h后基本入侵细胞。换新的培养液重新培养,每12 h观察一次,3～4 d出现第一代裂殖体,5～7 d出现第二代裂殖体,7～8 d出现卵囊。该模型为球虫有关生理生化、免疫学、疫苗制备、药物效力试验等方面的研究工作,以及球虫病的防治研究开辟了新途径。
(3)同时根据E.tenella HSP70基因(GenBank Locus:Z46965.1)设计特异性引物,以RT-PCR方法扩增了E. tenella广东株基因序列,并将其克隆至表达载体上,转入大肠埃希菌Rosetta菌,经IPTG诱导,实现其在宿主菌Rosetta中表达。先前表达的以pET-32a(+)为载体的HSP70蛋白多为包涵体形式存在于菌体细胞内,通过不断反复摸索表达条件,更换pMAL-c2X为表达载体,获得了可溶性的融合蛋白,并结合Amylose Resin层析获得较高浓度的目的蛋白。同时将HSP70基因插入pcDNA-6载体中,构建DNA疫苗表达载体,将构建好的质粒转染Vero细胞,结果表明,HSP70可以在细胞中成功表达。
(4)用ELISA以及鸡只抗球虫感染动物实验,分别检测和统计了鸡只体内重组蛋白免疫后相应抗体水平和盲肠病变记分、测定克盲肠内容物卵囊数(OPG)、增重、综合抗球虫指数(ACI)等相关抗球虫感染指标,证明重组蛋白免疫后能在机体内产生一定的抗体水平和抗球虫感染保护力。
Chicken coccidiosis is the parasitic protozoal disease caused by one of seven kinds of Eimeria alone or mixed in different parts of intestine.
(1)In this study,using chicken intestinal epithelial cells (IEC), instead of renal cell, a new type of chicken Eimeria culture model was established in vitro, because the condition and process of IEC infected with Eimeria is closer to that of the natural infection of chickens. The results of isolation and culture of chicken IEC in vitro showed that the chicken IEC adhered to the wall of plate after 24-48 hours, and formed into monolayer in 7～8 d, when inoculated to culture plate after digestion with typeⅠcollagenase 70 min in 37℃and low-speed centrifugation, and under the culture conditions of 5% CO2, 39.5℃, DMEM containing 5% fetal bovine serum.
(2)The purified sporozoites were collected, and inoculated to the primary embryo IEC. Using the culture model, the reproductive conditions of E.tenella schizony stage were explored, and the optimal medium and added components for E.tenella culture were determined, and the adaptive E.tenella was established in the chicken IEC. The results showed that sporozoites invaded into IEC in 2 hours when sporozoites were inoculated to the IEC cultured 8 days later. The medium was changed again, and the results weree observed once in every 12 hours. The first generation of schizonts were observed in 3-4 days, the second generation of schizonts were observed in 5-7 days, and the oocysts were appeared in 7-8 days post-inoculstion. The culture model in vitro laid new ways for the researches in the physiochemistry, immunology, vaccine preparation, medical effecacy, and the prevention and treatment of chicken coccidiosis.
(3)The specific primers were designed according to HSP70 gene, the gene sequence of E.tenella Guangdong strain (GenBank Locus: Z46965.1) was amplified, then the gene was cloned into expression vector, and transformed into E.coli Rosetta. The expression of protein in host bacteria was achieved with IPTG induction. The HSP70 protein expressed with vector PET-32a (+) was in the form of inclusion in the bacterial cells. The soluble fusion protein was expressed with vector pMAL-c2X after repeated determination of expression conditions. The higher concentration of protein was obtained by Amylose Resin chromatography. The HSP70 gene was inserted into vector pcDNA-6 to construct the DNA vaccine expression vector. The pcDNA-6-HSP70 plasmid was transfected into Vero cells. The results showed that HSP70 protein was successfully expressed in eukaryotic cells.
(4)The seral antibody titers of E. tenella infected chicken immunized with recombinant proteins were detected by ELISA. The parameters including cecal lesion score, oocysts per gram feces (OPG), weight gains, and anti-coccidial index (ACI) were observed. The results showed that the recombinant proteins induced certain degree antibody levels and anticoccidial immunoprotection.
(1)本研究利用鸡胚肠上皮细胞来替代鸡肾细胞,建立一种新型的鸡球虫体外细胞培养模型,因为球虫感染肠上皮细胞的条件和过程更加接近了鸡体自然感染状态。探讨了鸡胚肠上皮细胞体外分离培养的方法,结果显示,鸡胚肠上皮细胞,在37℃用Ⅰ型胶原酶分步消化70 min,低速离心后接种培养板,90 min后将未贴壁的细胞移至新的有胶原铺底的24孔培养板中,5%CO2 39.5℃,DMEM含5%胎牛血清的条件下培养,24～48 h细胞贴壁,7～8 d长成单层。
(2)收集纯化的球虫子孢子,接种于适合球虫生长发育的鸡胚肠上皮原代细胞,采用这一细胞模型探索了E.tenella裂殖生殖阶段的发育情况,经试验确定了最适球虫繁殖的培养基和添加成分,建立了艾美耳球虫鸡胚肠上皮细胞培养适应株。结果表明,在细胞培养第8天时接种子孢子,子孢子在接种2 h后基本入侵细胞。换新的培养液重新培养,每12 h观察一次,3～4 d出现第一代裂殖体,5～7 d出现第二代裂殖体,7～8 d出现卵囊。该模型为球虫有关生理生化、免疫学、疫苗制备、药物效力试验等方面的研究工作,以及球虫病的防治研究开辟了新途径。
(3)同时根据E.tenella HSP70基因(GenBank Locus:Z46965.1)设计特异性引物,以RT-PCR方法扩增了E. tenella广东株基因序列,并将其克隆至表达载体上,转入大肠埃希菌Rosetta菌,经IPTG诱导,实现其在宿主菌Rosetta中表达。先前表达的以pET-32a(+)为载体的HSP70蛋白多为包涵体形式存在于菌体细胞内,通过不断反复摸索表达条件,更换pMAL-c2X为表达载体,获得了可溶性的融合蛋白,并结合Amylose Resin层析获得较高浓度的目的蛋白。同时将HSP70基因插入pcDNA-6载体中,构建DNA疫苗表达载体,将构建好的质粒转染Vero细胞,结果表明,HSP70可以在细胞中成功表达。
(4)用ELISA以及鸡只抗球虫感染动物实验,分别检测和统计了鸡只体内重组蛋白免疫后相应抗体水平和盲肠病变记分、测定克盲肠内容物卵囊数(OPG)、增重、综合抗球虫指数(ACI)等相关抗球虫感染指标,证明重组蛋白免疫后能在机体内产生一定的抗体水平和抗球虫感染保护力。
Chicken coccidiosis is the parasitic protozoal disease caused by one of seven kinds of Eimeria alone or mixed in different parts of intestine.
(1)In this study,using chicken intestinal epithelial cells (IEC), instead of renal cell, a new type of chicken Eimeria culture model was established in vitro, because the condition and process of IEC infected with Eimeria is closer to that of the natural infection of chickens. The results of isolation and culture of chicken IEC in vitro showed that the chicken IEC adhered to the wall of plate after 24-48 hours, and formed into monolayer in 7～8 d, when inoculated to culture plate after digestion with typeⅠcollagenase 70 min in 37℃and low-speed centrifugation, and under the culture conditions of 5% CO2, 39.5℃, DMEM containing 5% fetal bovine serum.
(2)The purified sporozoites were collected, and inoculated to the primary embryo IEC. Using the culture model, the reproductive conditions of E.tenella schizony stage were explored, and the optimal medium and added components for E.tenella culture were determined, and the adaptive E.tenella was established in the chicken IEC. The results showed that sporozoites invaded into IEC in 2 hours when sporozoites were inoculated to the IEC cultured 8 days later. The medium was changed again, and the results weree observed once in every 12 hours. The first generation of schizonts were observed in 3-4 days, the second generation of schizonts were observed in 5-7 days, and the oocysts were appeared in 7-8 days post-inoculstion. The culture model in vitro laid new ways for the researches in the physiochemistry, immunology, vaccine preparation, medical effecacy, and the prevention and treatment of chicken coccidiosis.
(3)The specific primers were designed according to HSP70 gene, the gene sequence of E.tenella Guangdong strain (GenBank Locus: Z46965.1) was amplified, then the gene was cloned into expression vector, and transformed into E.coli Rosetta. The expression of protein in host bacteria was achieved with IPTG induction. The HSP70 protein expressed with vector PET-32a (+) was in the form of inclusion in the bacterial cells. The soluble fusion protein was expressed with vector pMAL-c2X after repeated determination of expression conditions. The higher concentration of protein was obtained by Amylose Resin chromatography. The HSP70 gene was inserted into vector pcDNA-6 to construct the DNA vaccine expression vector. The pcDNA-6-HSP70 plasmid was transfected into Vero cells. The results showed that HSP70 protein was successfully expressed in eukaryotic cells.
(4)The seral antibody titers of E. tenella infected chicken immunized with recombinant proteins were detected by ELISA. The parameters including cecal lesion score, oocysts per gram feces (OPG), weight gains, and anti-coccidial index (ACI) were observed. The results showed that the recombinant proteins induced certain degree antibody levels and anticoccidial immunoprotection.
引文
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