【摘要】： 目的:人乳頭瘤病毒(HPV)是一類感染表皮和粘膜鱗狀上皮的DNA病毒,其中,高危型HPV18與尖銳濕疣、宮頸上皮內(nèi)瘤以及其他上皮性腫瘤的發(fā)生相關(guān)。HPV18早期蛋白E7是特異性的腫瘤抗原,作為一種理想的靶抗原在免疫治療研究中倍受重視。本研究中,我們通過計算機輔助算法來篩選HPV18E7抗原的B細胞表位,并通過對其進行免疫學評價進一步鑒定預(yù)測的B細胞表位能否誘導(dǎo)抗原肽特異性抗體產(chǎn)生。為制備針對HPV18感染的治療性疫苗提供候選表位。方法:①計算機軟件預(yù)測。采用親水性方案、柔韌性方案、抗原指數(shù)方案及表面可能性方案對HPV18型E7抗原B細胞表位進行綜合評估,確定候選的B細胞表位。②預(yù)測肽合成、純化及質(zhì)譜鑒定。采用標準的Fmoc方案合成多肽,合成的多肽經(jīng)RP-HPLC純化及純度分析,并用質(zhì)譜進行定性鑒定。③表位多肽的免疫效果評價。實驗組分別以P1、P2、P3三條合成肽免疫Balb/c小鼠,陰性組以生理鹽水作為對照,與弗氏完全佐劑混合乳化后于0、2、4周在腋下、腹股溝及腹腔多點注射,于初次免疫后的1、3、5周,分別采集并制備各實驗組及陰性對照組小鼠血清。④收集人乳頭瘤病毒18型。收集新鮮宮頸癌標本1例(PCR法檢測HPV18陽性),將標本碾碎、超聲破膜,離心收集HPV病毒。⑤間接ELISA法進行鑒定。分別以合成肽段和宮頸癌組織上清液包被酶聯(lián)板,與采集的各實驗組小鼠血清結(jié)合,以陰性組小鼠血清作為對照。酶標二抗為羊抗小鼠IgG,底物為TMB【四甲基聯(lián)苯胺】。用酶標儀于492nm波長處測定吸光度A值。結(jié)果:①綜合各方案分析考慮:人乳頭瘤病毒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細胞優(yōu)勢表位。分別命名為P1、P2和P3。②多肽經(jīng)RP-HPLC純化及純度分析,合成肽的純度大于90%,經(jīng)質(zhì)譜分析合成肽的分子量測定值與理論值相符。③以三條多肽鏈包被酶聯(lián)板,與免疫后小鼠血清抗體結(jié)合反應(yīng)。初次免疫后第1周,實驗組小鼠血清抗體滴度均呈陰性;第3周,表位多肽P2、P3組均出現(xiàn)陽性,與陰性對照組具有顯著性差異(P0.05),P1組為陰性(P0.05);第5周,P1、P2、P3組均出現(xiàn)陽性結(jié)果,與陰性對照組均有顯著性差異(P0.05)。④以宮頸癌組織上清液包被酶聯(lián)板,只有P2、P3組產(chǎn)生的小鼠血清抗體與其結(jié)合反應(yīng)呈陽性(P0.05),表位肽P1產(chǎn)生的抗體不呈現(xiàn)陽性反應(yīng)(P0.05)。結(jié)論:①多種預(yù)測方案聯(lián)合應(yīng)用可提高預(yù)測效率,避免了在研究人乳頭瘤病毒18E7抗原表位時盲目合成多肽。②人乳頭瘤病毒18E729-44和人乳頭瘤病毒18E751-59具有較強的免疫原性,可能成為針對人乳頭瘤病毒18感染的治療性多肽疫苗的候選表位。
[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，

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