瑟氏泰勒虫p33基恩与牛IL-18基的串联表达
发布时间：2018-08-07 18:57
【摘要】：瑟氏泰勒虫(Theileria sergenti)是泰勒科、泰勒属的血液原虫,寄生在红细胞内。感染泰勒虫的牛主要表现为慢性贫血、高热、消瘦和淋巴结肿大等临床症状,感染泰勒虫的牛只由于贫血导致免疫力下降,极易引起其他血液性疾病的病原体的继发感染,进而导致死亡率的升高,给养牛产业造成不可避免的经济损失。近年来,随着中国牛养殖业的蓬勃发展,瑟氏泰勒虫的发病率在全国范围内的各个地区呈以惊人的速度呈上升趋势,给全国各地区的养牛产业造成巨大的经济威胁。因此,有效控制瑟氏泰勒虫的发病率成为当务之急。本试验将瑟氏泰勒虫主要表面抗原p33基因与牛白细胞介素18(IL-18)基因进行重组表达,结果表明表达出的重组蛋白IL-18-p33具有相当好的反应原性。瑟氏泰勒虫的p33基因是瑟氏泰勒虫的主要表面蛋白之一,能够引起宿主的良好的特异性免疫反应。根据GenBank上已经发表的p33基因序列[GenBank: DQ078264.1]白细胞介素18基因序列(IL-18) [GenBank:EU574909.1]分别设计两对特异性引物。提取感染瑟氏泰勒虫牛的阳性抗凝血液DNA,并以血液DNA为模板应用聚合酶链式反应技术(即PCR技术)扩增得到大小为821bp大小左右的目的片段。提取pMD-18-T-IL-18质粒DNA,以该质粒DNA为模板,通过PCR技术,得到大小为627 bp目的片段。应用SOE-PCR技术将两段基因串联,得到串联基因IL-18-p33,大小为1416 bp。将经SOP-PCR技术得到的串联基因片段与pMD-19-T simple载体连接,然后转化至大肠杆菌的克隆菌种DH5a。提取质粒DNA,质粒DNA经过PCR?BamH I, Xho I双酶切和测序确定串联基因IL-18-p33成功与pMD-19-T simple载体连接后,将该重组质粒命名为pMD-19T-IL18-p33。将质粒pMD-19T-IL18-p33和原核表达载体pGEX-4T1经过BamH I和Xho I双酶切,分别回收酶切后的IL18-p33和pGEX-4T1的目的条带,然后进行16℃过夜连接,并转化至大肠杆菌表达的菌株BL21,提取质粒DNA后进行质粒PCR鉴定和BanH I、 Xho I双酶切鉴定,并将其命名为BL21-pGEX4T1-IL18-p33。对BL21-pGEX4T1-IL18-p33进行1/1000浓度的IPTG诱导表达。将诱导产物进行SDS-PAGE凝胶电泳分析,结果在66.4 kDa与97.2 kDa之间出现一条79 kDa左右大小条带,且与预期结果大小相符。然后将表达的蛋白IL-18-p33进行Western blot分析,结果表明IL-18-p33蛋白具有相当好的反应原性。
[Abstract]:(Theileria sergenti) is a blood protozoa of the genus Thaleridae, which is parasitic in red blood cells. The main symptoms of Taylor's infection in cattle are chronic anemia, high fever, emaciation and enlarged lymph nodes. Cattle infected with Taylor's disease are susceptible to secondary infection by pathogens of other blood diseases because of anemia, which leads to a decrease in immunity. This leads to an increase in mortality and an inevitable economic loss to the cattle industry. In recent years, with the vigorous development of cattle farming in China, the incidence of Taylor serpentine is rising at an alarming rate in all regions of the country, which poses a huge economic threat to cattle farming in all regions of the country. Therefore, the effective control of the incidence of Taylor's disease has become a top priority. The recombinant expression of p33 gene and bovine interleukin 18 (IL-18) gene showed that the expressed recombinant protein IL-18-p33 was highly reactive. The p33 gene of Taylor serovar is one of the main surface proteins of Taylor serpentine, which can induce a good specific immune response of the host. Two pairs of specific primers were designed according to the published sequence of p33 gene [GenBank: DQ078264.1] interleukin-18 gene (IL-18) [GenBank:EU574909.1] from GenBank. The positive anticoagulant blood DNA was extracted from the infected cattle, and the target fragment was amplified by polymerase chain reaction (PCR) using blood DNA as template. The target fragment was about the size of 821bp. The pMD-18-T-IL-18 plasmid DNA was extracted and the target fragment of 627bp was obtained by PCR using the plasmid DNA as template. The tandem gene IL-18-p33, which was 1416 BP in size, was obtained by using SOE-PCR technique. The tandem gene fragment obtained by SOP-PCR technique was ligated with pMD-19-T simple vector and then transformed into E. coli clone strain DH 5a. After the plasmid PCR?BamH I, Xho I was digested and sequenced, the recombinant plasmid was named pMD-19T-IL18-p33 after the tandem gene IL-18-p33 was successfully ligated with the pMD-19-T simple vector. The plasmids pMD-19T-IL18-p33 and prokaryotic expression vector pGEX-4T1 were digested by BamH I and Xho I, and the target bands of IL18-p33 and pGEX-4T1 were recovered respectively, and then connected overnight at 16 鈩，
本文编号：2171041
[Abstract]:(Theileria sergenti) is a blood protozoa of the genus Thaleridae, which is parasitic in red blood cells. The main symptoms of Taylor's infection in cattle are chronic anemia, high fever, emaciation and enlarged lymph nodes. Cattle infected with Taylor's disease are susceptible to secondary infection by pathogens of other blood diseases because of anemia, which leads to a decrease in immunity. This leads to an increase in mortality and an inevitable economic loss to the cattle industry. In recent years, with the vigorous development of cattle farming in China, the incidence of Taylor serpentine is rising at an alarming rate in all regions of the country, which poses a huge economic threat to cattle farming in all regions of the country. Therefore, the effective control of the incidence of Taylor's disease has become a top priority. The recombinant expression of p33 gene and bovine interleukin 18 (IL-18) gene showed that the expressed recombinant protein IL-18-p33 was highly reactive. The p33 gene of Taylor serovar is one of the main surface proteins of Taylor serpentine, which can induce a good specific immune response of the host. Two pairs of specific primers were designed according to the published sequence of p33 gene [GenBank: DQ078264.1] interleukin-18 gene (IL-18) [GenBank:EU574909.1] from GenBank. The positive anticoagulant blood DNA was extracted from the infected cattle, and the target fragment was amplified by polymerase chain reaction (PCR) using blood DNA as template. The target fragment was about the size of 821bp. The pMD-18-T-IL-18 plasmid DNA was extracted and the target fragment of 627bp was obtained by PCR using the plasmid DNA as template. The tandem gene IL-18-p33, which was 1416 BP in size, was obtained by using SOE-PCR technique. The tandem gene fragment obtained by SOP-PCR technique was ligated with pMD-19-T simple vector and then transformed into E. coli clone strain DH 5a. After the plasmid PCR?BamH I, Xho I was digested and sequenced, the recombinant plasmid was named pMD-19T-IL18-p33 after the tandem gene IL-18-p33 was successfully ligated with the pMD-19-T simple vector. The plasmids pMD-19T-IL18-p33 and prokaryotic expression vector pGEX-4T1 were digested by BamH I and Xho I, and the target bands of IL18-p33 and pGEX-4T1 were recovered respectively, and then connected overnight at 16 鈩，
本文编号：2171041
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