HPV18型E7抗原B细胞表位的预测与鉴定
发布时间：2019-06-08 13:12
【摘要】： 目的:人乳头瘤病毒(HPV)是一类感染表皮和粘膜鳞状上皮的DNA病毒,其中,高危型HPV18与尖锐湿疣、宫颈上皮内瘤以及其他上皮性肿瘤的发生相关。HPV18早期蛋白E7是特异性的肿瘤抗原,作为一种理想的靶抗原在免疫治疗研究中倍受重视。本研究中,我们通过计算机辅助算法来筛选HPV18E7抗原的B细胞表位,并通过对其进行免疫学评价进一步鉴定预测的B细胞表位能否诱导抗原肽特异性抗体产生。为制备针对HPV18感染的治疗性疫苗提供候选表位。方法:①计算机软件预测。采用亲水性方案、柔韧性方案、抗原指数方案及表面可能性方案对HPV18型E7抗原B细胞表位进行综合评估,确定候选的B细胞表位。②预测肽合成、纯化及质谱鉴定。采用标准的Fmoc方案合成多肽,合成的多肽经RP-HPLC纯化及纯度分析,并用质谱进行定性鉴定。③表位多肽的免疫效果评价。实验组分别以P1、P2、P3三条合成肽免疫Balb/c小鼠,阴性组以生理盐水作为对照,与弗氏完全佐剂混合乳化后于0、2、4周在腋下、腹股沟及腹腔多点注射,于初次免疫后的1、3、5周,分别采集并制备各实验组及阴性对照组小鼠血清。④收集人乳头瘤病毒18型。收集新鲜宫颈癌标本1例(PCR法检测HPV18阳性),将标本碾碎、超声破膜,离心收集HPV病毒。⑤间接ELISA法进行鉴定。分别以合成肽段和宫颈癌组织上清液包被酶联板,与采集的各实验组小鼠血清结合,以阴性组小鼠血清作为对照。酶标二抗为羊抗小鼠IgG,底物为TMB【四甲基联苯胺】。用酶标仪于492nm波长处测定吸光度A值。结果:①综合各方案分析考虑:人乳头瘤病毒18E7蛋白的第15-22位,29-44位,51-59位,即E715-22(LEPQNEIP),E729-44(EQLSDSEEENDEIDGV),E751-59(ARRAEPQRH)可能是B细胞优势表位。分别命名为P1、P2和P3。②多肽经RP-HPLC纯化及纯度分析,合成肽的纯度大于90%,经质谱分析合成肽的分子量测定值与理论值相符。③以三条多肽链包被酶联板,与免疫后小鼠血清抗体结合反应。初次免疫后第1周,实验组小鼠血清抗体滴度均呈阴性;第3周,表位多肽P2、P3组均出现阳性,与阴性对照组具有显著性差异(P0.05),P1组为阴性(P0.05);第5周,P1、P2、P3组均出现阳性结果,与阴性对照组均有显著性差异(P0.05)。④以宫颈癌组织上清液包被酶联板,只有P2、P3组产生的小鼠血清抗体与其结合反应呈阳性(P0.05),表位肽P1产生的抗体不呈现阳性反应(P0.05)。结论:①多种预测方案联合应用可提高预测效率,避免了在研究人乳头瘤病毒18E7抗原表位时盲目合成多肽。②人乳头瘤病毒18E729-44和人乳头瘤病毒18E751-59具有较强的免疫原性,可能成为针对人乳头瘤病毒18感染的治疗性多肽疫苗的候选表位。
[Abstract]:Objective: human papillomavirus (HPV) is a class of DNA viruses that infect epidermis and mucous scaly epithelial cells, in which high risk HPV18 is associated with condyloma acuminatum. The occurrence of cervical intraepithelial tumors and other epithelial tumors is related. HPV 18 early protein E7 is a specific tumor antigen and has attracted much attention as an ideal target antigen in immunotherapy. In this study, we screened the B cell epitopes of HPV18E7 antigen by computer aided algorithm, and further identified whether the predicted B cell epitopes could induce the production of antigenic peptide specific antibodies by immunological evaluation. It provides candidate epitopes for the preparation of therapeutic vaccines against HPV18 infection. Methods: 1 computer software prediction. The B cell epitopes of HPV 18 E7 antigen were comprehensively evaluated by hydrophilic scheme, flexibility scheme, antigen index scheme and surface possibility scheme, and the candidate B cell epitopes were determined. 2 Predictive peptide synthesis, purification and mass spectrometry identification. The peptides were synthesized by standard Fmoc scheme. The peptides were purified and purified by RP-HPLC and identified qualitatively by mass spectrometry. The immune effect of 3 epitope peptides was evaluated. Balb/c mice were immunized with P _ 1, P _ 2 and P _ 3 synthetic peptides, respectively. In the negative group, saline was used as control and emulsified with Freund's complete adjuvant at 0, 2, 4 weeks after multiple injection in axillary, inguinal and abdominal cavity. The serum of mice in each experimental group and negative control group was collected and prepared at 1, 3 and 5 weeks after the first immunization. 4 Human papillomavirus type 18 was collected. One case of fresh cervical cancer (positive for HPV18 detected by PCR) was collected. The specimens were crushed, broken by ultrasound, and HPV virus was collected by centrifugation. 5 indirect ELISA method was used for identification. The synthetic peptide and cervical cancer tissue culture medium were coated with enzyme-linked plate and bound to the serum of each experimental group, and the serum of negative group was used as control. The substrate of sheep anti-mouse IgG, was TMB [tetramethylbenzidine]. The absorbance A value was determined by enzyme labeling instrument at 492nm wavelength. Results: 1 the 15th to 22nd position, 29 鈮，
本文编号：2495307
[Abstract]:Objective: human papillomavirus (HPV) is a class of DNA viruses that infect epidermis and mucous scaly epithelial cells, in which high risk HPV18 is associated with condyloma acuminatum. The occurrence of cervical intraepithelial tumors and other epithelial tumors is related. HPV 18 early protein E7 is a specific tumor antigen and has attracted much attention as an ideal target antigen in immunotherapy. In this study, we screened the B cell epitopes of HPV18E7 antigen by computer aided algorithm, and further identified whether the predicted B cell epitopes could induce the production of antigenic peptide specific antibodies by immunological evaluation. It provides candidate epitopes for the preparation of therapeutic vaccines against HPV18 infection. Methods: 1 computer software prediction. The B cell epitopes of HPV 18 E7 antigen were comprehensively evaluated by hydrophilic scheme, flexibility scheme, antigen index scheme and surface possibility scheme, and the candidate B cell epitopes were determined. 2 Predictive peptide synthesis, purification and mass spectrometry identification. The peptides were synthesized by standard Fmoc scheme. The peptides were purified and purified by RP-HPLC and identified qualitatively by mass spectrometry. The immune effect of 3 epitope peptides was evaluated. Balb/c mice were immunized with P _ 1, P _ 2 and P _ 3 synthetic peptides, respectively. In the negative group, saline was used as control and emulsified with Freund's complete adjuvant at 0, 2, 4 weeks after multiple injection in axillary, inguinal and abdominal cavity. The serum of mice in each experimental group and negative control group was collected and prepared at 1, 3 and 5 weeks after the first immunization. 4 Human papillomavirus type 18 was collected. One case of fresh cervical cancer (positive for HPV18 detected by PCR) was collected. The specimens were crushed, broken by ultrasound, and HPV virus was collected by centrifugation. 5 indirect ELISA method was used for identification. The synthetic peptide and cervical cancer tissue culture medium were coated with enzyme-linked plate and bound to the serum of each experimental group, and the serum of negative group was used as control. The substrate of sheep anti-mouse IgG, was TMB [tetramethylbenzidine]. The absorbance A value was determined by enzyme labeling instrument at 492nm wavelength. Results: 1 the 15th to 22nd position, 29 鈮，
本文编号：2495307
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