【作者】 高全胜；

【导师】 黄复生；

【作者基本信息】 第三军医大学 ， 病原生物学， 2006， 博士

【摘要】 恶性肿瘤严重威胁着人类健康,探寻抗恶性肿瘤的新策略一直是基础与临床研究的热点和难点。APRIL亦TALL-2(TNF and apoptosis ligand-related leukocyte-expressedligand 2),于1998年被发现和克隆,属TNF超家族新成员,细胞表面的APRIL经furin转换酶裂解,其部分胞外区从膜上脱落,成为天然sAPRIL(可溶型,即105-250氨基酸区域),其生物学活性与全长(即膜结合型)APRIL基本一致,研究表明105-250氨基酸为人APRIL发挥功能的主要区域。近年研究表明,在多种恶性肿瘤细胞增殖、存活以及促肿瘤形成过程中,APRIL过表达均发挥独特作用,故有效抑制该分子表达或功能活性,可望为抗肿瘤治疗提供新策略。Dalum等人提出,经异源T辅助细胞(T helper cell,Th)表位修饰的自身突变细胞因子,可作为具有交叉反应性的新抗原,诱生内源性抗天然细胞因子抗体。该理论在抗TNF-α的实验研究中得到证实。基于此,本研究开展如下工作:①克隆编码人天然sAPRIL的cDNA,以此为模板,经PCR分别克隆缺失不同区域的sAPRIL cDNA突变体(msAPRIL cDNA);②将HEL Th表位的DNA序列重组于各msAPRIL cDNA的5′端或3′端,然后将重组DNA分别克隆于原核表达载体(本项目选用pQE-80L),制备由msAPRIL和HEL Th表位组成的融合蛋白;③将融合蛋白纯化,并证实相应融合蛋白已丧失正常sAPRIL的功能活性;④用各融合蛋白分别免疫正常小鼠,使融合蛋白交叉诱生抗人sAPRIL抗体,并观察其对人sAPRIL促肿瘤细胞增殖的抑制作用;⑤以人外周血单个核细胞重建裸鼠免疫功能,用能诱导出较高滴度抗人sAPRIL抗体的融合蛋白免疫该裸鼠,观察相应融合蛋白交叉诱生抗人sAPRIL抗体的作用,以及相应抗体对人sAPRIL促肿瘤细胞增殖的抑制作用,并初步探索该策略对白血病小鼠的治疗作用。本课题获得如下结果:1.构建了编码人sAPRIL的cDNA(aa105-aa250)借助RT-PCR,从人外周血单个核细胞中将其克隆,将其构建于pQE-80L,序列分析表明与GenBank所报道编码sAPRIL的cDNA序列完全一致。2.构建了经HEL Th表位修饰、缺失不同区域的sAPRIL cDNA突变体(msAPRILcDNA)借助PCR,以pQE-80L/sAPRIL为模板,分别克隆缺失不同区域的sAPRIL cDNA突变体,通过核酸连接技术在相应缺失端加入HEL Th表位。3.构建了突变体原核表达载体将经HEL Th表位修饰、缺失不同区域的sAPRIL cDNA突变体分别克隆于原核表达载体pQE-80L,测序证实序列正确。4.制备了融合蛋白在大肠杆菌中诱导sAPRIL及各融合蛋白表达,并借助Western blot鉴定表达产物。5.纯化了蛋白产物表达产物经SDS-PAGE检测,主要以包涵体形式存在。用Ni-NTA柱纯化包涵体溶解物,目的蛋白洗脱液经多步透析法复性后冷冻抽干,获得一定量各种纯化蛋白。6.筛选出不具有sAPRIL生物活性的突变体蛋白借助MTT法证实:N、C端均连接HEL Th表位的重组sAPRIL突变融合蛋白,其在体外能刺激Raji细胞增殖;经HEL Th表位修饰、N/C端缺失45bp(简称C、D)或90bp(简称E、F)的sAPRIL突变融合蛋白,其在体外不能刺激Raji细胞增殖。由此表明,已成功制备不具活性的sAPRIL突变体C、D、E和F,且HEL Th表位加入并不影响sAPRIL活性。7.探讨了不具生物活性的突变融合蛋白诱导小鼠产生多克隆抗体的作用将C、D、E和F纯化蛋白加佐剂后免疫小鼠,经ELISA和Western blot鉴定,融合蛋白能刺激小鼠产生多克隆抗体,同时获得抗C、D、E和F的抗血清。8.探讨了抗sAPRIL突变体多抗对sAPRIL生物活性的影响采用MTT检测发现:抗C、D、E和F抗血清可分别抑制sAPRIL促Raji和Jurkat细胞增殖作用(但抗F抗血清对sAPRIL促Raji细胞增殖作用无影响),其中以抗D抗血清抑制效果最强。9.探讨了突变体D及其抗血清的生物学作用用突变体D免疫无渗漏的SCID小鼠,采用ELISA和Western blot鉴定抗体产生,MTT检测该多克隆抗体对sAPRIL促Raji和Jurkat细胞增殖的影响。结果发现:成功获得抗人sAPRIL的多克隆交叉抗体;该多克隆抗体能抑制sAPRIL促Raji和Jurkat细胞增殖作用。初步表明:已筛选出基于增殖诱导配体的新型抗瘤效应分子。10.验证了突变体D纯化蛋白对白血病动物的疗效体外试验证实:sAPRIL呈剂量依赖性促进P388白血病细胞增殖;突变体D丧失刺激P388白血病细胞增殖的活性;用突变体D纯化蛋白干预P388诱导的白血病小鼠,能延长其生存期,但参照T/C值(评价药物抗瘤作用的常用指标)该治疗无效。更多还原

【Abstract】 Malignant tumors are a great threat to human health, and to seek effective molecules and appropriate methods to counteract them has long been an effort of researchers. APRIL, also called TALL-2 (TNF and apoptosis ligand-related leukocyte-expressed ligand 2), was discovered and cloned in 1998 as a new member of the TNF superfamily. The expressed APRIL is cleaved by furin convertase, and part of the extracellular domain falls off the membrane, resulting in the formation of natural sAPRIL (soluble APRIL, i.e., amino acids 105-250). Full-length (i.e., membrane-bound) APRIL and sAPRIL are basically of the same biological activity. It was shown that amino acids 105-250 constitute the main active domain of human APRIL. Recent studies have also demonstrated that overexpression of APRIL plays a peculiar role in the proliferation and survival of malignant tumor cells as well as in the promotion of tumorigenesis. To effectively inhibit the functional activity of APRIL may provide a new method to treat relevant tumors.The present study was based on the status of research of APRIL-inhibiting molecules, the hypothesis formulated by Dalum et al who held that mutant cytokines modified by heterologous T helper cell (Th) epitopes may act as new antigens inducing endogenous, cross-reactive antibodies against natural cytokines, and precise evidence obtained from studies related to anti-TNF-a. In the present study, cDNA encoding natural human sAPRIL was cloned and used as template to clone sAPRIL cDNA mutants (msAPRIL cDNA) with deletion of different domains by PCR. Then HEL Th epitope-encoding DNA sequences were recombined with 5’- or 3 ’-terminal of msAPRIL cDNA, and recombinant DNA was cloned into prokaryotic expression vectors pQE-80L to express the fusion protein composed of msAPRIL and HEL Th epitope. Fusion proteins were purified, and were confirmed to lose the functional activity of normal sAPRIL. Fusion proteins were used to immunize normal mice, and the induction of cross-reactive anti-human sAPRIL antibody by fusion proteins and the inhibition of the antibodies on the enhancement of tumor cell proliferation by human sAPRIL were observed. Fusion proteins that may induce high titres of anti-human sAPRIL antibodies were used to immunize nude mice, the immunity of which was reestablished by human peripheral blood mononuclear cells. The induction of cross-reactive anti-human sAPRIL antibodies by fusion proteins, the inhibition of the antibodies on the enhancement of tumor cell proliferation by human sAPRIL and the therapeutic effect of the antibodies on leukemia mice were observed.Based on the analyses above, we carried out the study in attempt to seek novel candidate molecules with multiple advantages for immunotherapy (even prevention of recurrence) of APRIL-related tumors. The main results are as follows:1. Human sAPRIL-encoding cDNA (aa105-aa250) was cloned by RT-PCR from human peripheral blood nucleated cells and constructed into pQE-80L. Sequence analysis confirmed that the sequence of the cloned sAPRIL cDNA was identical to that registered in GenBank.2. We cloned sAPRIL cDNA mutants (msAPRIL cDNA) with deletion of various domains by PCR using pQE-80L/sAPRIL as template. HEL Th epitope modified sAPRIL cDNA mutants were obtained by linking HEL Th epitopes to the deleted ends using nucleic acid ligation technique.3. The resulting HEL Th epitope modified sAPRIL cDNA mutants were cloned into the prokaryotic express vector pQE-80L, and sequence analysis confirmed the sequences of the obtained prokaryotic express vectors carrying cDNA mutants were correct.4. To obtain fusion proteins, we induced the expression of sAPRIL and fusion proteins in E. coli. Western blot confirmed the expression of target fusion proteins.5. To obtain purified proteins, the expressed proteins were subjected to SDS-PAGE which confirmed that the proteins mainly located in inclusion bodies. Lysates of inclusion bodies were purified by using Ni-NTA column, and the target protein eluent was subjected to renaturation by step-wise dialysis and lyophilization to obtain the purified proteins.6. To screen mutant proteins with no biological activity of sAPRIL, the MTT assay was used. The results showed that recombinant sAPRIL and mutant fusion proteins with HEL Th epitopes at both N- and C-terminals can stimulate in vitro proliferation of Raji cells, while mutant sAPRIL modified by HEL Th epitopes with deletions at the N- and C-terminals of 45bp (C, D) or 90bp deletion (E, F) cannot. The results demonstrated that mutants C, D, E and F had no sAPRIL activity and HEL Th epitopes did not affect sAPRIL activity.7. To test whether mutant proteins with no biological activity induce polyclonal antibodies in mice, mice were immunized by purified proteins C, D, E and F and immunoadjuvant. ELISA and Western blot analyses confirmed that the fusion proteins may induce polyclonal antibodies in mice. Meanwhile, antisera against proteins C, D, E and F were obtained.8. To clarify the effect of polyclonal antibodies on the biological activity of sAPRIL, the inhibition of polyclonal antibodies on Raji and Jurkat cell proliferation stimulated by sAPRIL was assessed by the MTT assay. The results indicated that the antisera may inhibit Raji and Jurkat cell proliferation stimulated by sAPRIL, however, antiserum against protein F did not inhibit Raji cell proliferation stimulated by sAPRIL. Among the four antisera, the antiserum against mutant protein D was the strongest in inhibiting the proliferation of the two kinds of cells.9. To verify whether mutant protein D may induce cross-reactive human sAPRIL polyclonal antibodies in SCID mice leaky phenotype and the influence of these polyclonal antibodies on Raji and Jurkat cell proliferation stimulated by sAPRIL, we immunized SCID mice leaky phenotype with mutant D, identified the resulting antibodies by ELISA and Western blot, and assessed by the MTT assay the effect of these polyclonal antibodies on Raji and Jurkat cell proliferation stimulated by sAPRIL. The results indicated that cross-reactive human sAPRIL polyclonal antibodies were obtained successfully, which may inhibit Raji and Jurkat cell proliferation stimulated by sAPRIL. The results preliminarily demonstrated that we have screened novel candidate molecules for proliferation inducing ligand-based tumor immunotherapy.10. To clarify the efficacy of the purified mutant protein D in leukemia animal models, we first assayed the influence of sAPRIL and mutant protein D on the proliferation of P388 leukemic cells. The results indicated that sAPRIL may promote the proliferation of P388 leukemic cells in a dose-dependent manner. Mutant protein D stimulated the proliferative activity of P388 leukemic cells, and prolonged the survival of mice with P388 induced leukemia; however, the therapy was ineffective in light of T/C value, a common parameter to evaluate the efficacy of a drug for tumor.更多还原