【摘要】：瑟氏泰勒蟲(Theileria sergenti)是泰勒科、泰勒屬的血液原蟲,寄生在紅細(xì)胞內(nèi)。感染泰勒蟲的牛主要表現(xiàn)為慢性貧血、高熱、消瘦和淋巴結(jié)腫大等臨床癥狀,感染泰勒蟲的牛只由于貧血導(dǎo)致免疫力下降,極易引起其他血液性疾病的病原體的繼發(fā)感染,進(jìn)而導(dǎo)致死亡率的升高,給養(yǎng)牛產(chǎn)業(yè)造成不可避免的經(jīng)濟(jì)損失。近年來,隨著中國牛養(yǎng)殖業(yè)的蓬勃發(fā)展,瑟氏泰勒蟲的發(fā)病率在全國范圍內(nèi)的各個地區(qū)呈以驚人的速度呈上升趨勢,給全國各地區(qū)的養(yǎng)牛產(chǎn)業(yè)造成巨大的經(jīng)濟(jì)威脅。因此,有效控制瑟氏泰勒蟲的發(fā)病率成為當(dāng)務(wù)之急。本試驗(yàn)將瑟氏泰勒蟲主要表面抗原p33基因與牛白細(xì)胞介素18(IL-18)基因進(jìn)行重組表達(dá),結(jié)果表明表達(dá)出的重組蛋白IL-18-p33具有相當(dāng)好的反應(yīng)原性。瑟氏泰勒蟲的p33基因是瑟氏泰勒蟲的主要表面蛋白之一,能夠引起宿主的良好的特異性免疫反應(yīng)。根據(jù)GenBank上已經(jīng)發(fā)表的p33基因序列[GenBank: DQ078264.1]白細(xì)胞介素18基因序列(IL-18) [GenBank:EU574909.1]分別設(shè)計(jì)兩對特異性引物。提取感染瑟氏泰勒蟲牛的陽性抗凝血液DNA,并以血液DNA為模板應(yīng)用聚合酶鏈?zhǔn)椒磻?yīng)技術(shù)(即PCR技術(shù))擴(kuò)增得到大小為821bp大小左右的目的片段。提取pMD-18-T-IL-18質(zhì)粒DNA,以該質(zhì)粒DNA為模板,通過PCR技術(shù),得到大小為627 bp目的片段。應(yīng)用SOE-PCR技術(shù)將兩段基因串聯(lián),得到串聯(lián)基因IL-18-p33,大小為1416 bp。將經(jīng)SOP-PCR技術(shù)得到的串聯(lián)基因片段與pMD-19-T simple載體連接,然后轉(zhuǎn)化至大腸桿菌的克隆菌種DH5a。提取質(zhì)粒DNA,質(zhì)粒DNA經(jīng)過PCR?BamH I, Xho I雙酶切和測序確定串聯(lián)基因IL-18-p33成功與pMD-19-T simple載體連接后,將該重組質(zhì)粒命名為pMD-19T-IL18-p33。將質(zhì)粒pMD-19T-IL18-p33和原核表達(dá)載體pGEX-4T1經(jīng)過BamH I和Xho I雙酶切,分別回收酶切后的IL18-p33和pGEX-4T1的目的條帶,然后進(jìn)行16℃過夜連接,并轉(zhuǎn)化至大腸桿菌表達(dá)的菌株BL21,提取質(zhì)粒DNA后進(jìn)行質(zhì)粒PCR鑒定和BanH I、 Xho I雙酶切鑒定,并將其命名為BL21-pGEX4T1-IL18-p33。對BL21-pGEX4T1-IL18-p33進(jìn)行1/1000濃度的IPTG誘導(dǎo)表達(dá)。將誘導(dǎo)產(chǎn)物進(jìn)行SDS-PAGE凝膠電泳分析,結(jié)果在66.4 kDa與97.2 kDa之間出現(xiàn)一條79 kDa左右大小條帶,且與預(yù)期結(jié)果大小相符。然后將表達(dá)的蛋白IL-18-p33進(jìn)行Western blot分析,結(jié)果表明IL-18-p33蛋白具有相當(dāng)好的反應(yīng)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，

本文編號：2171041