人源抗狂犬病毒磷蛋白基因工程抗體及細胞內抗體的研究
發(fā)布時間：2018-11-02 16:17
【摘要】：本研究采用Fab噬菌體載體pComb 3H系統(tǒng),選擇了5位體內含高滴度的抗狂犬病毒抗體的志愿者,采集他們的抗凝外周血并分離淋巴細胞,提取細胞內的總RNA,并逆轉錄為互補的cDNA,利用人Fab抗體引物擴增抗體輕鏈和重鏈Fd段因片段,分別通過輕鏈酶切位點Sac I/Xba I和重鏈鏈酶切位點Xho I/Spe I克隆入pComb 3H噬菌體抗體庫載體,構建成人源抗狂犬病毒的噬菌體Fab抗體庫。將Fab抗體庫菌庫包裝成噬菌體庫,使用純化的狂犬病毒顆粒aG株和CTN株對噬菌體庫進行富集篩選,在最后一輪篩選富集后,挑取每個庫至少挑取200個克隆進行原核誘導表達,包被抗人Fab抗體檢測Fab抗體表達上清進行抗體表達檢測,包被病毒顆粒檢測抗體結合的特異性,雙陽性克隆序列測定獲得序列,登錄VBASE2網(wǎng)站,輸入抗體序列,通過與數(shù)據(jù)庫中序列比較,確定抗體的輕鏈亞類和重鏈類型,以及可變區(qū)的CDR區(qū)基因。我們獲得2株重鏈相同輕鏈相似的Fab抗體,選擇其中一株表達為IgG全抗體,通過ELISA.免疫印跡和間接免疫熒光實驗證實抗體特異性結合磷蛋白。雖然中和試驗表明磷蛋白抗體并沒有中和病毒的作用,但磷蛋白可以結合核蛋白、聚合酶蛋白,在調節(jié)病毒轉錄和復制過程發(fā)揮作用。分析磷蛋白序列,利用截短突變的方法確定表位,以狂犬病病毒磷蛋白全長為模板,N端截短,C段不變,構建P1,P2,P3,P4,P5截短克隆；C端截短,N段不變,每隔20個氨基酸進行截短,構建△C20,△C40,△C60,△C80,△C100截短克隆,截短片段C端帶有HIS標簽,瞬時轉染表達,WB分析表位。結果顯示,磷蛋白截短克隆P1、P2、P3、P4、P5和P△C20、P△C40、P△C60、P△C80、P △C100與抗HIS抗體反應,說明磷蛋白截短蛋白均有表達；而只有P1,P2,P3, P4,P5和P△C20與RV1A2反應,P△C40,P△C60,P△C80,P△C100卻沒有反應,提示目的表位位于257～277位氨基酸之間。隨后構建更短截短片段的截短突變,將表位定于262～266位氨基酸(VLGWV)之間。查詢磷蛋白晶體結構(186～297)(PDB：1VYI),分析磷蛋白3D結構和抗原抗體對接,推測RV1A2結合W265氨基酸。分析狂犬病毒屬7種基因型的129條序列,發(fā)現(xiàn)該位點(VLGWV)高度保守。將RV1A2抗體改造為單鏈抗體(scFv),克隆入真核細胞表達載體pEF/myc/cyto中,在細胞質內定位表達,保證80%以上的細胞均有陽性表達。用狂犬病病毒攻擊毒株CVS-11進行染毒,染毒后一到四天每天鑒定細胞內病毒復制情況和上清中病毒分泌情況。研究顯示,細胞內抗體具有明顯的病毒復制抑制功能,抗磷蛋白細胞內抗體在病毒易感細胞MNA細胞的表達會降低上清中的病毒滴度。由于抗體表位位于C端,與P1, P2, P3, P4, P5均能結合,具有較好的的結合廣譜,而且該位點高度保守,因此狂犬病病毒磷蛋白C端會是一個較好的作用靶點。然而細胞內抗體抑制病毒增殖只是初步試驗,還需要更進一步證據(jù)。細胞內抗體作為一種藥物靶點,如何穿過血腦屏障和精確的細胞內定位,還有很長的路要走。
[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.
【學位授予單位】：中國疾病預防控制中心
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R392.11
本文編號：2306308
[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.
【學位授予單位】：中國疾病預防控制中心
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R392.11
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