【摘要】： 豬囊尾蚴病是全球性寄生性蠕蟲病,發(fā)展中國(guó)家更為常見,尤其在我國(guó),該病的流行給人民生產(chǎn)和生活帶來諸多危害,給人類的健康帶來嚴(yán)重的影響。我國(guó)至少已有31個(gè)省(市、自治區(qū))有過該病的報(bào)道,囊尾蚴病患者約1000萬,每年屠宰的囊尾蚴病豬約1200萬頭,直接經(jīng)濟(jì)損失約20億元。腦囊尾蚴病對(duì)人類的健康危害極大,在某些發(fā)展中國(guó)家,腦囊尾蚴病是引起人死亡的主要原因之一。由于抗藥性的不斷出現(xiàn)以及藥物殘留等問題的嚴(yán)重性,使得利用免疫預(yù)防技術(shù)來取代藥物成為控制豬囊尾蚴病的必然。免疫毒素是一種導(dǎo)向藥物,它將毒素基因和靶向基因融合,表達(dá)融合蛋白,具有識(shí)別和選擇性地破壞受體靶細(xì)胞的功能,它由導(dǎo)向部分和毒性部分組成。綠膿桿菌外毒素通常作為重組毒素的毒性成分,其毒性本質(zhì)是它能夠催化真核細(xì)胞延伸因子的ADP核糖基化,阻止肽鏈延長(zhǎng)并中斷細(xì)胞蛋白質(zhì)的合成而行使細(xì)胞毒作用。作為重組毒素的導(dǎo)向部分,除能夠與特定靶細(xì)胞表面受體結(jié)合外,同時(shí)能夠與毒素基因相融合,表達(dá)出融合蛋白,且二者不影響各自獨(dú)立結(jié)構(gòu)和功能。將豬囊尾蚴頭節(jié)單鏈抗體基因與綠膿桿菌外毒素PE40基因連接,裝入表達(dá)載體pET-28(a)中,以獲得重組毒素,研究其對(duì)豬囊尾蚴的殺滅作用。 從綠膿桿菌ATCC27853菌株中提取基因組,根據(jù)GenBank發(fā)表的綠膿桿菌外毒素基因全序列,自行設(shè)計(jì)引物,分別引入XbaⅠ和HindⅢ酶切位點(diǎn)。采用PCR方法擴(kuò)增PE40基因,在反應(yīng)體系中加入10%總體積的甘油,電泳檢測(cè),純化的PCR產(chǎn)物與pMD18-T載體連接構(gòu)建質(zhì)粒pMD18-T-PE40,轉(zhuǎn)入大腸桿菌感受態(tài)JM109中,藍(lán)白斑篩選陽性克隆菌,提取質(zhì)粒進(jìn)行酶切鑒定及PCR鑒定,并測(cè)序。質(zhì)粒pMD18-T-PE40經(jīng)XbaⅠ和HindⅢ雙酶切后電泳顯示2692bp和1083bp兩條帶,測(cè)序結(jié)果顯示擴(kuò)增的PE40基因共有10個(gè)堿基發(fā)生改變,變異的堿基引起7個(gè)氨基酸發(fā)生變化,但PE40肽段的重要活性位點(diǎn)與綠膿桿菌標(biāo)準(zhǔn)產(chǎn)毒株P(guān)A103相比均未發(fā)生變化。含有ScFv基因的質(zhì)粒pMD18-T-ScFv已經(jīng)構(gòu)建,且ScFv基因兩端的酶切位點(diǎn)分別為EcoRⅠ和XbaⅠ,用XbaⅠ和HindⅢ分別對(duì)質(zhì)粒pMD18-T-PE40和pMD18-T-ScFv進(jìn)行雙酶切,利用XbaⅠ和HindⅢ酶切位點(diǎn)將PE40基因與質(zhì)粒pMD18-T-ScFv相連接,轉(zhuǎn)入大腸桿菌感受態(tài)JM109中,挑取陽性菌酶切鑒定。經(jīng)酶切電泳分析,得到大小約為1800bp的目的片段,與預(yù)期結(jié)果相同,說明ScFv與PE40已連接成功。用EcoRⅠ和HindⅢ分別對(duì)重組質(zhì)粒pMD18-T-ScFv-PE40和表達(dá)載體pET-28(a)進(jìn)行酶切,將ScFv-PE40轉(zhuǎn)入pET-28(a)表達(dá)載體中構(gòu)建融合毒素pET28-ScFv-PE40,轉(zhuǎn)入大腸桿菌感受態(tài)細(xì)胞DH5α中,挑取陽性菌落提取質(zhì)粒,酶切電泳檢測(cè),得到目的條帶大小約為1800bp,保存菌種,為進(jìn)一步研究重組免疫毒素對(duì)豬囊尾蚴的殺滅作用奠定基礎(chǔ)。
[Abstract]:Cysticercosis is a global parasitic worm disease, which is more common in developing countries. Especially in China, the epidemic of cysticercosis has brought a lot of harm to people's production and life, and brought serious impact to human health. At least 31 provinces (municipalities and autonomous regions) have reported the disease. About 10 million cysticercosis patients and 12 million cysticercosis pigs slaughtered each year with direct economic loss of 2 billion yuan. Cerebral cysticercosis is harmful to human health. In some developing countries, cerebral cysticercosis is one of the main causes of death. Due to the continuous emergence of drug resistance and the severity of drug residues, the use of immunoprophylaxis to replace drugs has become a necessity for the control of cysticercosis. Immunotoxin is a kind of targeted drug, which fuses toxin gene and target gene, expresses fusion protein, has the function of recognizing and selectively destroying receptor target cells, and it consists of directed and toxic parts. Pseudomonas aeruginosa exotoxin is usually used as the toxic component of recombinant toxin. Its toxic essence is that it can catalyze the ADP ribosylation of eukaryotic cell extension factor, prevent peptide chain from prolonging and interrupt the synthesis of cellular protein. In addition to binding to the surface receptors of specific target cells, the recombinant toxin can be fused with the toxin gene to express the fusion protein, and they do not affect their independent structures and functions. The single chain antibody gene of cysticercus cephalosporium was linked with the PE40 gene of Pseudomonas aeruginosa and inserted into the expression vector pET-28 (a) to obtain the recombinant toxin and to study its killing effect on cysticercus cellulosae. The genome was extracted from Pseudomonas aeruginosa ATCC27853 strain. According to the whole sequence of exotoxin gene of Pseudomonas aeruginosa published by GenBank, primers were designed to introduce Xba 鈪，

本文編號(hào)：2211274