人源抗狂犬病毒磷蛋白基因工程抗体及细胞内抗体的研究

发布时间：2018-11-02 16:17

【摘要】：本研究采用Fab噬菌体载体pComb 3H系统,选择了5位体内含高滴度的抗狂犬病毒抗体的志愿者,采集他们的抗凝外周血并分离淋巴细胞,提取细胞内的总RNA,并逆转录为互补的cDNA,利用人Fab抗体引物扩增抗体轻链和重链Fd段因片段,分别通过轻链酶切位点Sac I/Xba I和重链链酶切位点Xho I/Spe I克隆入pComb 3H噬菌体抗体库载体,构建成人源抗狂犬病毒的噬菌体Fab抗体库。将Fab抗体库菌库包装成噬菌体库,使用纯化的狂犬病毒颗粒aG株和CTN株对噬菌体库进行富集筛选,在最后一轮筛选富集后,挑取每个库至少挑取200个克隆进行原核诱导表达,包被抗人Fab抗体检测Fab抗体表达上清进行抗体表达检测,包被病毒颗粒检测抗体结合的特异性,双阳性克隆序列测定获得序列,登录VBASE2网站,输入抗体序列,通过与数据库中序列比较,确定抗体的轻链亚类和重链类型,以及可变区的CDR区基因。我们获得2株重链相同轻链相似的Fab抗体,选择其中一株表达为IgG全抗体,通过ELISA.免疫印迹和间接免疫荧光实验证实抗体特异性结合磷蛋白。虽然中和试验表明磷蛋白抗体并没有中和病毒的作用,但磷蛋白可以结合核蛋白、聚合酶蛋白,在调节病毒转录和复制过程发挥作用。分析磷蛋白序列,利用截短突变的方法确定表位,以狂犬病病毒磷蛋白全长为模板,N端截短,C段不变,构建P1,P2,P3,P4,P5截短克隆；C端截短,N段不变,每隔20个氨基酸进行截短,构建△C20,△C40,△C60,△C80,△C100截短克隆,截短片段C端带有HIS标签,瞬时转染表达,WB分析表位。结果显示,磷蛋白截短克隆P1、P2、P3、P4、P5和P△C20、P△C40、P△C60、P△C80、P △C100与抗HIS抗体反应,说明磷蛋白截短蛋白均有表达；而只有P1,P2,P3, P4,P5和P△C20与RV1A2反应,P△C40,P△C60,P△C80,P△C100却没有反应,提示目的表位位于257～277位氨基酸之间。随后构建更短截短片段的截短突变,将表位定于262～266位氨基酸(VLGWV)之间。查询磷蛋白晶体结构(186～297)(PDB：1VYI),分析磷蛋白3D结构和抗原抗体对接,推测RV1A2结合W265氨基酸。分析狂犬病毒属7种基因型的129条序列,发现该位点(VLGWV)高度保守。将RV1A2抗体改造为单链抗体(scFv),克隆入真核细胞表达载体pEF/myc/cyto中,在细胞质内定位表达,保证80%以上的细胞均有阳性表达。用狂犬病病毒攻击毒株CVS-11进行染毒,染毒后一到四天每天鉴定细胞内病毒复制情况和上清中病毒分泌情况。研究显示,细胞内抗体具有明显的病毒复制抑制功能,抗磷蛋白细胞内抗体在病毒易感细胞MNA细胞的表达会降低上清中的病毒滴度。由于抗体表位位于C端,与P1, P2, P3, P4, P5均能结合,具有较好的的结合广谱,而且该位点高度保守,因此狂犬病病毒磷蛋白C端会是一个较好的作用靶点。然而细胞内抗体抑制病毒增殖只是初步试验,还需要更进一步证据。细胞内抗体作为一种药物靶点,如何穿过血脑屏障和精确的细胞内定位,还有很长的路要走。
[Abstract]:In this study, the Fab phage vector pComb 3H system was used to select the volunteers with anti-HAV antibody with high drop in 5 sites, collect their anti-coagulated peripheral blood and separate lymphocytes, extract the total RNA in the cells, and reverse transcription into complementary cDNA. A human Fab antibody primer was used to amplify the antibody light chain and the heavy chain Fd fragment, and the fragment was cloned into the pComb 3H phage antibody library vector by the light chain restriction site Saac I/ Xba I and the heavy chain restriction enzyme site Xho I/ Spe I, respectively, and the phage Fab antibody library of the adult human Fab antibody library was constructed. packaging the Fab antibody library bacteria library into a phage library, enriching and screening the phage library by using the purified cryolite particle aG strain and the CTN strain, selecting at least 200 clones of each bank for prokaryotic induction expression after the last round of screening and enrichment, coating anti-human Fab antibody detection Fab antibody expression supernatant for antibody expression detection, coating virus particle detection antibody binding specificity, double positive clone sequence determination obtaining sequence, logging in VBASE2 website, inputting antibody sequence, comparing with sequence in database, The light chain sub-class and the heavy chain type of the antibody, and the CDR region gene of the variable region are determined. We obtained a Fab antibody similar to the same light chain of two heavy chains, one of which was expressed as an IgG whole antibody, by ELISA. Immunoblotting and indirect immunofluorescence assay confirmed antibody-specific binding of phosphoproteins. Although neutralization tests show that the phosphoprotein antibody is not neutralizing the virus, the phosphoprotein can bind to the nucleoprotein, the polymerase protein, and play a role in regulating viral transcription and replication. analyzing the phosphoprotein sequence, determining the table position by using a truncated mutation method, wherein the whole length of the rabies virus phosphorus protein is a template, the N end is cut short, the C section is unchanged, and P1, P2, P3, P4 and P5 are cut short and cloned; the C end is cut short, the N section is unchanged, the length of every 20 amino acids is cut short, and the CDC20 is constructed, C100 truncated clones were truncated, and the C-end of truncated segment C had HIS label, transient transfection expression and WB analysis table. The results showed that the phosphorus protein truncated clones P1, P2, P3, P4, P5 and P, C20, P, C40, P, C60, P, C80, P, C100 were reacted with the anti-HIS antibody, indicating that the phosphoprotein truncated proteins were all expressed; and only P1, P2, P3, P4, P5 and P-C20 were reacted with the RV1A2, P-type C40, P, C60, P, C80, P, C100 did not respond, suggesting that the target table was located between 257 and 277 amino acids. A truncated mutation of shorter truncated segments is then constructed and the table bits are set to be between 262 and 266 amino acids (VLGWV). The phosphoprotein crystal structure (186-297) (Accession No. 1VYI) was queried, and the binding of the phosphoprotein 3D structure with the antigen antibody was analyzed, and the putative RV1A2 binding to the W265 amino acid. 129 sequences of 7 genotypes were analyzed, and the site (VLGWV) was found to be highly conserved. The RV1A2 antibody was transformed into single chain antibody (scFv), cloned into eukaryotic expression vector pEF/ myc/ cyto, and expressed in cytoplasm. The strain CVS-11 was infected with rabies virus, and the virus replication and virus secretion were identified every day from one to four days after exposure. The study shows that the intracellular antibody has obvious virus replication inhibitory function, and anti-phosphorus-protein intracellular antibody can decrease the virus drop in the supernatant of virus-susceptible cell MNA cells. Because the antibody table bit is located at the C terminal, it can bind to P1, P2, P3, P4 and P5, has better binding broad spectrum, and the site is highly conserved, so the rabies virus phosphoprotein C terminal can be a better action target. however, that intracellular antibody inhibit viral proliferation is only a preliminary test and further evidence is needed. The intracellular antibody as a drug target, how to cross the blood-brain barrier and accurate intracellular localization, and a long way to go.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R392.11

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