【摘要】： 炭疽是由炭疽芽孢桿菌(Bacillus anthracis)引起的一種急性、烈性人畜共患病,因其發(fā)病率高、潛伏期短、致死率高、炭疽芽孢對環(huán)境具有極強抵抗力等特點而使炭疽芽孢桿菌常被用做生物武器或生物恐怖戰(zhàn)劑。青霉素等抗生素藥物是治療炭疽的傳統(tǒng)方法,但其缺點顯著。治療性抗體(中和抗體)是感染后唯一有效的治療藥物,其中人源性中和抗體由于療效肯定、特異性高、毒副作用小而被公認(rèn)為最理想的抗體形式,因此研究開發(fā)治療炭疽的人源性中和抗體藥物具有重要意義。 根據(jù)炭疽毒素的致病機制,保護性抗原的第四結(jié)構(gòu)域(簡稱PAD4)與細(xì)胞受體結(jié)合后介導(dǎo)了水腫因子和致死因子進入細(xì)胞內(nèi)發(fā)揮毒素作用,PAD4在炭疽毒素致病中起著基礎(chǔ)性的作用。因此本研究將炭疽毒素PAD4進行原核表達和純化,以重組蛋白作為抗原表位,利用噬菌體抗體庫技術(shù)制備抗炭疽桿菌PAD4抗體,為研制治療炭疽的人源性中和抗體藥物奠定基礎(chǔ)。 本研究依照炭疽桿菌PAD4的氨基酸序列(NCBI中的P13423序列,140aa)和基因序列(420bp,GenBank Accession:AY428556)設(shè)計并優(yōu)化其核苷酸序列,在化學(xué)合成12條寡核苷酸片段的基礎(chǔ)上,利用重疊延伸多聚酶鏈?zhǔn)椒磻?yīng)(SOE-PCR)合成PAD4基因,克隆入表達載體中實現(xiàn)了PAD4與輔助噬菌體M13KO7GⅢ蛋白N1結(jié)構(gòu)域的可溶性融合表達和純化。其次,以高純度的融合蛋白為靶抗原從全合成人源性噬菌體抗體庫(庫容為5×109)中篩選具有特異性的人源性噬菌體單鏈抗體,實現(xiàn)人源性抗體基因在大腸桿菌中的可溶性表達并鑒定表達產(chǎn)物與PAD4的特異性結(jié)合活性。 本研究采用重疊延伸PCR反應(yīng)合成了PAD4基因,實現(xiàn)了大腸桿菌的高水平可溶性表達,表達產(chǎn)物約占細(xì)菌總蛋白量的36%,經(jīng)親和層析純化獲得了純度達到電泳級以上的重組蛋白。經(jīng)過篩選獲得了四株與炭疽毒素PAD4特異性結(jié)合的人源性單鏈抗體AP、APD17、APD22和APDC。 由于篩選的噬菌體抗體APDC輕鏈CDR3中含有琥珀突變密碼子TAG,因此設(shè)計并合成了兩條引物,進行重疊延伸PCR將TAG定點突變?yōu)镃AG。將改造的噬菌體抗體APDC基因和其余三株噬菌體抗體AP、APD17、APD22基因分別克隆入表達載體PTIG-TRX-E中,最終實現(xiàn)了四株抗體在大腸桿菌中的可溶性表達,經(jīng)初步鑒定大腸桿菌表達的四株抗體AP、APD17、APD22和APDC能與炭疽毒素PAD4特異性結(jié)合。
[Abstract]:Anthrax is an acute, acute zoonosis caused by Bacillus anthracis (Bacillus anthracis), because of its high incidence, short incubation period and high mortality. Because of its strong resistance to environment, Bacillus anthracis is often used as biological weapon or bioterrorist warfare agent. Antibiotic drugs such as penicillin are traditional methods for anthrax treatment, but their disadvantages are obvious. Therapeutic antibody (neutralizing antibody) is the only effective therapeutic drug after infection. Human neutralizing antibody is recognized as the most ideal form of antibody because of its positive curative effect, high specificity and little side effects. Therefore, it is of great significance to study and develop human neutralizing antibody drugs for anthrax treatment. According to the pathogenicity mechanism of anthrax toxin, the fourth domain of protective antigen (PAD4) binds to cell receptor and mediates the entry of edema factor and lethal factor into the cell to play the role of toxin. PAD4 plays a fundamental role in the pathogenesis of anthrax toxin. Therefore, the anthrax toxin PAD4 was expressed and purified in prokaryotic expression and purified, and the recombinant protein was used as antigen epitope to prepare PAD4 antibody against Bacillus anthracis by phage antibody library technology, which laid a foundation for the development of human neutralizing antibody drug for anthrax treatment. In this study, the nucleotide sequences were designed and optimized according to the amino acid sequence (P13423, 140aa) and gene sequence (420 BP PAD4 Accession:AY428556) of Bacillus anthracis PAD4, and 12 oligonucleotide fragments were chemically synthesized. PAD4 gene was synthesized by overlapping extension polymerase chain reaction (SOE-PCR) and cloned into expression vector. The soluble fusion expression and purification of PAD4 and N1 domain of auxiliary phage M13KO7G 鈪，

本文編號：2408960