【摘要】：瑟氏泰勒蟲(Theileriasergenti)是寄生于牛體內(nèi)的一種血液原蟲,是泰勒蟲科、泰勒蟲屬,主要寄生于牛的淋巴細(xì)胞、紅細(xì)胞、巨噬細(xì)胞中,通過(guò)蜱進(jìn)行傳播�，F(xiàn)階段,隨著養(yǎng)牛業(yè)的不斷發(fā)展,牛的瑟氏泰勒蟲病近幾年發(fā)生頻率呈現(xiàn)上升趨勢(shì),對(duì)養(yǎng)牛業(yè)的危害極大。但到目前為止還沒(méi)有特效藥物用于瑟氏泰勒蟲病的治療和預(yù)防,而且尚無(wú)該病的疫苗。瑟氏泰勒蟲p33基因,是該蟲體的主要表面蛋白基因,具有較好的免疫原性和反應(yīng)原性,認(rèn)為是較理想的候選抗原。真核表達(dá)系統(tǒng),除了具備原核表達(dá)系統(tǒng)的優(yōu)點(diǎn)外,還具有表達(dá)外源蛋白量高、遺傳性狀穩(wěn)定、產(chǎn)物提純簡(jiǎn)單等諸多優(yōu)點(diǎn),受到越來(lái)越多的人青睞。本試驗(yàn)根據(jù)Genbank中T.sergentip33基因中國(guó)株序列,設(shè)計(jì)一對(duì)特異性引物,用常規(guī)PCR擴(kuò)增出大小為786 bp的p33目的基因片段,將其克隆到pMD-19-T載體上,構(gòu)建pMD-19-T-p33重組克隆質(zhì)粒,經(jīng)PCR、雙酶切鑒定測(cè)序分析,證實(shí)目的片段正確地克隆到pMD-19-T載體上。然后將p33基因亞克隆到酵母表達(dá)載體pPICZaA上,重組質(zhì)粒經(jīng)PCR、EcoRⅠ和XbaⅠ雙酶切鑒定,證實(shí)成功構(gòu)建含有目的基因片段的真核表達(dá)載體pPICZα A-p33。然后將其轉(zhuǎn)化到GS115酵母感受態(tài)細(xì)胞中,最終通過(guò)800μg/ml濃度的博來(lái)霉素篩選得到高拷貝重組子,經(jīng)PCR篩選陽(yáng)性菌株后,通過(guò)最終濃度為1%的甲醇誘導(dǎo)并在畢赤酵母中大量表達(dá)外源蛋白,然后對(duì)誘導(dǎo)產(chǎn)物進(jìn)行SDS-PAGE,得到大小為29.67Ku的蛋白,與預(yù)期結(jié)果相符合。而后對(duì)誘導(dǎo)蛋白進(jìn)行Western blot反應(yīng)原性鑒定,蛋白可以被牛瑟氏泰勒蟲的陽(yáng)性血清特異性識(shí)別,表明得到的蛋白具有較好的反應(yīng)原性。本試驗(yàn)結(jié)果為以后瑟氏泰勒蟲病診斷試劑盒、瑟氏泰勒蟲p33基因的單克隆抗體和瑟氏泰勒蟲病疫苗研究等研究打下堅(jiān)實(shí)的基礎(chǔ)。
[Abstract]:(Theileriasergenti) is a blood protozoa parasitic in cattle. It is mainly parasitic on bovine lymphocytes, erythrocytes and macrophages, and is transmitted by ticks. At present, with the continuous development of cattle industry, the frequency of Taylor's disease in cattle is increasing in recent years, which is harmful to cattle industry. But so far there is no specific drug for the treatment and prevention of Taylor's disease, and there is no vaccine for the disease. The p33 gene is the main surface protein gene of the worm, and has good immunogenicity and reactivity, so it is considered to be an ideal candidate antigen. Eukaryotic expression system not only has the advantages of prokaryotic expression system but also has many advantages such as high expression amount of exogenous protein stable hereditary character simple product purification and so on. According to the Chinese strain sequence of T.sergentip33 gene in Genbank, a pair of specific primers were designed. The p33 gene fragment of 786 bp was amplified by conventional PCR and cloned into pMD-19-T vector to construct the recombinant pMD-19-T-p33 clone plasmid. The target fragment was correctly cloned into pMD-19-T vector by PCR, double digestion and sequencing analysis. Then the p33 gene was subcloned into yeast expression vector pPICZaA, and the recombinant plasmid was identified by PCR,EcoR 鈪，

本文編號(hào)：2266092