表达前列腺特异性膜抗原的DNA疫苗对肿瘤细胞的抑制作用

目的构建表达前列腺特异性膜抗原（PSMA）的DNA疫苗，观察其在体外对肿瘤细胞的免疫攻击和在体内对肿瘤细胞攻击的免疫保护作用。方法通过稳定转染构建表达PSMA的小鼠黑色素瘤细胞系B16-PSMA，将DNA疫苗pCDNA3．1-PSMA通过肌肉注射导入C57BL／6小鼠体内，分离小鼠脾细胞，检测细胞毒性T淋巴细胞（cytotoxic T lymphocytes，CTL）反应。以B16-PSMA细胞攻击免骺小鼠，观察免疫动物的无瘤生存期和肿瘤体积增长情况，评价DNA疫苗的抗肿瘤作用。结果DNA疫苗可诱导小鼠脾淋巴细胞CTL活性，经过免疫后的小鼠成瘤率降低，无瘤生存期延长，肿瘤生长缓慢，肿瘤组织内有较多淋巴细胞浸润，表明产生较强的抗肿瘤反应。结论表达PSMA的DNA疫苗能够诱导小鼠产生特异性免疫反应，对表达PSMA的肿瘤细胞的攻击产生免疫保护作用，为前列腺癌的预防和免疫治疗提供了新的思路。     

人β防御素-2与前列腺癌特异性膜抗原嵌合蛋白真核表达质粒构建及其诱导的小鼠特异性免疫应答

探索人β防御素2 (h BD- 2 )和前列腺特异性膜抗原(PSMA)共表达重组核酸疫苗针对前列腺癌的免疫治疗。研究以pc DNA3.1为载体,构建重组质粒pc DNA3.1/PSMA和pc DNA3.1/h BD- 2 - PSMA,通过RT- PCR和免疫组化检测其表达。免疫小鼠后,进行血清中抗体检测,CD4 、CD8 T淋巴细胞数目测定及CTL 特异性杀伤作用检测。结果显示构建的质粒转染COS- 7细胞后能表达目的基因,免疫小鼠后能在体内持久表达,可以诱导产生特异性抗体,能有效的刺激T细胞增生,诱导特异性CTL 反应。当以h BD- 2作为免疫佐剂时,CTL 活性更强。本研究成功的构建了含PSMA的表达质粒,免疫小鼠可以诱导出有效的体液和细胞免疫,为前列腺癌的免疫治疗奠定了一定的实验基础     

PSMA基因疫苗抑瘤效应及免疫机制的实验研究

目的： 观察表达前列腺特异性膜抗原(PSMA)基因疫苗在荷瘤小鼠模型中抑瘤效应并探讨其免疫机制,为前列腺癌的预防和免疫治疗提供实验基础。方法: 将PSMA基因疫苗肌注入BALB/c小鼠体内,检测其血清中PSMA抗体水平并观察脾T细胞的增殖效应和杀细胞毒效应,以sp2/0-PSMA细胞攻击免疫后小鼠,观察小鼠的成瘤率、肿瘤大小、平均瘤重及生存率,评价PSMA基因疫苗的抑瘤效应。结果: PSMA基因疫苗可诱导实验组小鼠产生PSMA抗体,脾T细胞的增殖效应和杀细胞毒效应,在一定时间内随着免疫次数的增加和时间的延长,抗体水平、脾细胞的增殖和杀细胞毒效应均呈上升趋势。与对照组相比较,实验组小鼠成瘤率低,无瘤生存期延长,肿瘤生长缓慢。结论: PSMA基因疫苗能诱导实验组小鼠产生特异性体液及细胞免疫应答,且有明显的抑瘤效应。     

基因疫苗pcDNA3．0．PSMA肿瘤细胞模型的建立

目的 构建表达前列腺特异性膜抗原（PSMA）的肿瘤细胞模型，为基因疫苗的抑瘤效应研究及免疫机制的探讨提供实验材料。方法 脂质体转染法分别将PSMA．pcDNA3．0质粒和pcDNA3．0质粒转染至SP2／0细胞,G418筛选后获得了稳定生长的阳性克隆细胞株。RT．PCR、间接免疫荧光法、Westernblot检测PSMA蛋白的表达。结果 RT．PCR、间接免疫荧光法和Westernblot均证明转染了PSMA．pcDNA3．0质粒的SP2／0细胞表达PSMA。结论 成功建立了稳定表达PSMA的小鼠肿瘤细胞模型．最终为前列腺癌的预防和免疫治疗研究打下了坚实的基础。     

布氏杆菌pCDNA3.1-L7/L12核酸疫苗的构建及其免疫学评价

目的 获得布氏杆菌保护性抗原L2／L12重组蛋白及pCDNA3．1-L7／L12重组质粒，并比较其诱导特异性免疫应答的能力。方法 PCR扩增布氏杆菌核蛋白L7／L12基因分别构建至原核表达载体PET32a( )和真核表达载体pCDNA3.1( )中；pET32a-L7／L12重组质粒转化BL21(DE3)，所表达蛋白经SDS-PAGE、免疫印迹分析、纯化后免疫小鼠；pCDNA3.1-12／L12重组质粒配以GM-CSF同时肌肉注射免疫小鼠，3次免疫后测定免疫功能进行免疫效果的评价。结果ELISA、Western blot检测到免疫鼠体内有特异性抗体产生，蛋白苗所诱导的抗体效价远远高于DNA疫苗；通过淋巴细胞增殖实验、细胞因子和CD分子测定表明DNA疫苗以诱发TH1型免疫为主。结论 所构建的布氏杆菌DNA疫苗和蛋白苗均具有诱导特异性细胞和体液免疫应答的能力，DNA疫苗诱导产生的细胞免疫反应强于蛋白苗，可作为潜在的布氏菌新型疫苗，有进一步研究的意义。     

The Proinflammatory Cytokine, IL-6, and its Interference with bFGF Signaling and PSMA in Prostate Cancer Cells

The aim of the present work was to study the expression of the proinflammatory cytokine, interleukin-6 (IL-6), mediated by bFGF signaling and its possible crosstalk with prostate-specific membrane antigen (PSMA) in LNCaP and PC3-PSMA prostate cancer cell lines. PC3 cells stably transfected with PSMA gene were used for restoring PSMA expression. LNCaP and PC3-PSMA cells were exposed to 10 ng/mL of basic fibroblast growth factor (bFGF). IL-6 production was measured by ELISA assay, and levels of PSMA expression were assessed by flow cytometry. AKT, ERK1/2, and p38 phosphorylation were detected by Western blot. bFGF enhances IL-6 production in LNCaP and PC3-PSMA prostate cancer cells. The effect of bFGF on stimulating IL-6 secretion was greater in LNCaP than in PC3-PSMA cells. In the presence of bFGF, PSMA expression was activated after 4 days of treatment in LNCaP and PC3-PSMA cells. This activation was not maintained after long term of treatment in both metastatic cell lines. Solely MAPKs pathways (ERK1/2 and p38) were activated after bFGF stimulation in both metastatic cell lines, whereas AKT did not show any activation. The interference of the proinflammatory cytokine, IL-6, with bFGF signaling and PSMA, should be of high clinical relevance in the treatment of metastatic prostate cancer. In developing novel therapeutic modalities targeting IL-6, significant attention should be given to PSMA and its inactivation to fight against prostate cancer.     

Biodegradable CpG DNA hydrogels for sustained delivery of doxorubicin and immunostimulatory signals in tumor-bearing mice

Immunostimulatory CpG DNA was self-assembled to form DNA hydrogels for use as a sustained delivery system for both intercalated doxorubicin (DXR) and immunostimulatory CpG motifs for cancer treatment. X-shaped DNA (X-DNA) was designed as a building unit, and underwent ligation to form DNA hydrogels. Two types of X-DNA were constructed using four oligodeoxynucleotides each, one containing six potent CpG motifs (CpG X-DNA) and the other with none (CpG-free X-DNA). CpG X-DNA was more effective than its components or the CpG-free counterpart in terms of the production of tumor necrosis factor-α from murine macrophage-like RAW264.7 cells, as well as maturation of the murine dendritic DC2.4 cells. The cytotoxic effects of X-DNA, DXR and their complexes were examined in a co-culture system of colon26/Luc cells, a murine adenocarcinoma clone stably expressing firefly luciferase, and RAW264.7 cells. DXR/CpG X-DNA showed the highest ability to inhibit the proliferation of colon26/Luc cells. DXR was slowly released from CpG DNA hydrogels. Injections of DXR/CpG DNA hydrogels into a subcutaneous colon26 tumor effectively inhibited tumor growth. These results show that CpG DNA hydrogels are an effective sustained system for delivery of immunostimulatory signals to TLR9-positive immune cells and DXR to cancer cells.     

载体和基因片段的选择对戊型肝炎病毒基因免疫抗原表达的影响

目的比较不同载体和不同大小的目的基因片段对戊型肝炎病毒(HEV)基因免疫抗原表达的影响,为基于HEV中和抗原表位的HEV基因免疫提供一定的依据。方法将含Ⅳ型HEV中国株中和抗原表位的p166和p179片段编码基因分别克隆入pTR421和pCDNA3.1两种真核表达载体,用脂质体介导基因转染HepG2人肝癌细胞系,经间接免疫荧光和Western blot分析以及将质粒注射小鼠局部肌肉组织后经免疫组织化学染色检测,分析目的基因在体内外的表达水平。结果成功构建了pTR421-166、pTR421-179、pCDNA3.1-166和pCDNA3.1-179四种重组质粒,经限制性内切酶双酶切和核苷酸测序鉴定,编码基因正确无误;pTR421-179转染的细胞以及注射小鼠的局部肌肉组织可以检测到p179的表达,而pCDNA3.1-179、pCDNA3.1-166以及pTR421-166均检测不到目的基因在体内、外的抗原表达。结论载体和目的基因片段的选择显著影响HEV抗原在体内、外的表达,直接关系到基因免疫的成功与否。     

The role of interleukine-12 in protection induced by CpG ODN against Listeria monocytogenes in BALB/c and C57BL/6

Synthetic oligodeoxynucleotides containing CpG motifs have several immune effects such as cytokine production in normal mice. In this study, we demonstrated the protective effect of CpG ODN against Listeria monocytogenes in BALB/c and C57BL/6. With a single dose of 40 μg/mouse of CpG ODN 48 h before bacterial challenge, protection was achieved in both strains of mice based on survival rates compared with controls. Serum IL-12 from each mouse was measured by using enzyme-linked immunosorbent assay (ELISA), at day 0 (48 h after CpG treatment) and at days 5, 11, and 15 after bacterial challenge. It was shown that serum IL-12 was only elevated at day 0 in BALB/c mice. However, for C57BL/6 mice, IL-12 was elevated at days 0, 5, and 11. These data support the hypothesis that CpG DNA motifs activate protective innate immune defenses.     

Combined dendritic cell- and CpG oligonucleotide-based immune therapy cures large murine tumors that resist chemotherapy

The use of dendritic cells (DC) loaded with tumor antigen is one of the most advanced approaches in cancer immunotherapy. CpG motifs within microbial DNA detected by toll-like receptor 9 are responsible for the favorable properties of CpG oligodeoxynucleotides (ODN) as immune modulators. In this study, mature antigen-pulsed DC or peritumoral injections of CpG ODN, both effective for the treatment of small established tumors, were almost ineffective against large established tumors (1-cm diameter) in a syngeneic murine colon carcinoma model. For large tumors, the antitumor activity of mature antigen-pulsed DC was strongly increased by coinjection of CpG ODN, resulting in a transient control of tumor growth. Rejection of large tumors and long-term cure of mice was achieved by combining injection of antigen-pulsed DC plus CpG ODN at a site distant to the tumor with peritumoral injections of CpG ODN. Depletion of CD8 T cells abrogated the therapeutic activity. Large numbers of DEC-205-positive DC infiltrated the tumor in treated mice. Therapy with 5-fluorouracil and leucovorin was unable to control tumors of the same size. In conclusion, we demonstrate that the immune system, provided that appropriate stimulation with DC and CpG ODN is given, has the potential to cure animals of large solid tumors in situations where even chemotherapy is not efficient.     

CpG oligodeoxynucleotides and interleukin-12 improve the efficacy of Mycobacterium bovis BCG vaccination in mice challenged with M. tuberculosis

Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only vaccine approved for prevention of tuberculosis. It has been postulated that serial passage of BCG over the years may have resulted in attenuation of its effectiveness. Because interleukin-12 (IL-12) and oligodeoxynucleotides (ODN) containing cytidine phosphate guanosine (CpG) motifs have been shown to enhance Th1 responses in vivo, they were chosen as adjuvants to increase the effectiveness of BCG vaccination. In this report, mice were vaccinated with BCG with or without IL-12 or CpG ODN and then challenged 6 weeks later via the aerosol route with the Erdman strain of M. tuberculosis. Mice vaccinated with BCG alone showed a 1- to 2-log reduction in bacterial load compared with control mice that did not receive any vaccination prior to M. tuberculosis challenge. Moreover, the bacterial loads of mice vaccinated with BCG plus IL-12 or CpG ODN were a further two- to fivefold lower than those of mice vaccinated with BCG alone. As an immune correlate, the antigen-specific production IFN-gamma and mRNA expression in spleen cells prior to challenge were evaluated. Mice vaccinated with BCG plus IL-12 or CpG ODN showed enhanced production of IFN-gamma compared with mice vaccinated with BCG alone. Finally, granulomas in BCG-vaccinated mice were smaller and more lymphocyte rich than those in unvaccinated mice; however, the addition of IL-12 or CpG ODN to BCG vaccination did not alter granuloma formation or result in added pulmonary damage. These observations support a role for immune adjuvants given with BCG vaccination to enhance its biologic efficacy.     

T helper 2 immunity to hepatitis B surface antigen primed by gene-gun-mediated DNA vaccination can be shifted towards T helper 1 immunity by codelivery of CpG motif-containing oligodeoxynucleotides

Gene-gun-mediated DNA immunization usually induces predominant T helper 2 (Th2) type immune response. As oligodeoxynucleotides (ODN)-containing unmethylated CpG motifs can activate the innate immune system in a Th1-biased way, the potential of codelivery of CpG motifs-containing ODN (CpG-ODN) with plasmid DNA to switch the gene-gun-mediated Th2 immune response was evaluated in this study. Here we show that codelivery of CpG-ODN with plasmid DNA at certain ratio (10/1) can enhance the Th1 humoral and cell-mediated immune responses in gene-gun-mediated DNA immunization in BALB/c mice, including increasing the hepatitis B surface antigen-specific total immunoglobulin G (IgG), IgG2a subclass, cytotoxic T-cell lymphocyte activity as well as interferon-gamma (IFN-gamma) secretion. Taken together, these results demonstrate that codelivery of CpG-ODN with recombinant plasmid DNA by gene gun can shift the gene-gun-mediated DNA immune response from Th2 towards Th1.     

A live Leishmania major vaccine containing CpG motifs induces the de novo generation of Th17 cells in C57BL/6 mice

Cutaneous leishmaniasis produces open sores that lead to scarring and disfiguration. We have reported that vaccination of C57BL/6 mice with live Leishmania major plus CpG DNA (Lm/CpG) prevents lesion development and provides long‐term immunity. Our current study aims to characterize the components of the adaptive immune response that are unique to Lm/CpG. We find that this vaccine enhances the proliferation of CD4⁺ Th17 cells, which contrasts with the highly polarized Th1 response caused by L. major alone; the Th17 response is dependent upon release of vaccine‐induced IL‐6. Neutralization of IFN‐γ and, in particular, IL‐17 caused increased parasite burdens in Lm/CpG‐vaccinated mice. IL‐17R‐deficient Lm/CpG‐vaccinated mice develop lesions, and display decreased IL‐17 and IFN‐γ, despite normal IL‐12, production. Neutrophil accumulation is also decreased in the IL‐17R‐deficient Lm/CpG‐vaccinated mice but Treg numbers are augmented. Our data demonstrate that activation of immune cells through CpG DNA, in the presence of live L. major, causes the specific induction of Th17 cells, which enhances the development of a protective cellular immunity against the parasite. Our study also demonstrates that vaccines combining live pathogens with immunomodulatory molecules may strikingly modify the natural immune response to infection in an alternative manner to that induced by killed or subunit vaccines.     

插入IL-2基因优化HBV DNA疫苗的研究

目的：观察IL-2基因的插入对DNA疫苗免疫效应的影响，探讨优化DNA疫苗设计、提高DNA疫苗兔疫效应的途径。方法：采用PCR产物直接克隆和重组DNA技术构建了人IL-2和HBsAg融合基因的真核表达载体pcDNA3．1 -S/IL-2，通过脂质体基因转移技术导入Cos-7细胞中检测其瞬间表达，并经肌肉注射免疫C57Bl/6小鼠，以检测和比较它们的细胞和体液免疫应答。结果：通过酶切、PCR及测序证实已正确完整地插入 IL-2基因，成功构建了 pcDNA3．1 -S/IL-2重组质粒。体外转染Cos-7后可见基因的表达和分泌，pcDNA3．1 -S/IL-2可以提高免疫小鼠的抗 HBs抗体滴度和脾淋巴细胞诱生的IL-2的生物活性水平，增加 HBsAg特异性的脾淋巴细胞增殖指数。结论：IL-2和 HBsAg基因的融合表达对 DNA疫苗的免疫反应起协同和增强作用，提示IL-2基因插入可能是增强DNA疫苗的免疫效应的可行途径。     

DNA fusion gene vaccines induce cytotoxic T-cell attack on naturally processed peptides of human prostate-specific membrane antigen

For long-term attack on tumor cells in patients with prostate cancer, induction of cytolytic T cells is desirable. Several lineage-specific target proteins are known and algorithms have identified candidate MHC class I-binding peptides, particularly for HLA-A*0201. We have designed tolerance-breaking DNA fusion vaccines incorporating a domain of tetanus toxin fused to candidate tumor-derived peptide sequences. Using three separate peptide sequences from prostate-specific membrane antigen (PSMA) (peptides PSMA(27) , PSMA(663) , and PSMA(711) ), this vaccine design induced high levels of CD8(+) T cells against each peptide in a HLA-A(*) 0201 preclinical model. In contrast, the full-length PSMA sequence containing all three epitopes was poorly immunogenic. Induced T cells were cytotoxic against peptide-loaded tumor cells, but only those against PSMA(27) or PSMA(663) peptides, and not PSMA(711) , were able to kill tumor cells expressing endogenous PSMA. Cytotoxicity was also evident in vivo. The preclinical model provides a powerful tool for generating CD8(+) T cells able to predict whether target cells can process and present peptides, essential for planning peptide vaccine-based clinical trials.     

Plasmid DNA vaccines are effective in the absence of IFNgamma.

Intramuscular injection of bacterially derived plasmid DNA results in the development of both humoral and cellular immune responses against plasmid-encoded antigens. Immunostimulatory CpG sequences within bacterial DNA are thought to enhance this process by stimulating the secretion of proinflammatory cytokines such as interferon gamma (IFNgamma) by cells of the innate immune system. Although IFNgamma induction by CpG elements within plasmid DNA has been documented in vitro and more recently in vivo, and coimmunization with plasmids expressing IFNgamma has been shown to enhance DNA-immunization-induced immune responses, it is unclear if IFNgamma is necessary for successful DNA immunization. To address this issue, we compared humoral and cellular immune responses in wild-type and IFNgamma-deficient mice vaccinated with a plasmid (pCMVNP) expressing the nucleoprotein gene from the arenavirus lymphocytic choriomeningitis virus (LCMV). IFNgamma-positive (BALB/c) and IFNgamma-negative (GKO) mice responded to DNA vaccination by the development of antigen-specific CD8(+) T cells, which were detectable directly ex vivo by intracellular cytokine staining and comprised 0.7-2.5% of all CD8(+) T cells in the vaccine. DNA vaccines also induced virus-specific cytotoxic T lymphocytes (CTL), even in the absence of IFNgamma. DNA vaccination of both mouse strains also was associated with a significant reduction in viral titers after LCMV challenge, indicating that, at least in the presence of other immune effector mechanisms, IFNgamma is not required for induction of protective anti-viral immunity by DNA immunization. No quantitative differences were observed in antiviral IgG levels among GKO and BALB/c vaccinees, although GKO mice did exhibit a significant reduction of the IgG2a:IgG1 ratio, in agreement with the previously documented requirement for IFNgamma in isotype switching to IgG2a. Immunized BALB/c mice produced similar levels of both IgG1 and IgG2a, indicating a mixed Th1/Th2 response to intramuscular immunization with pCMVNP. These results show that IFNgamma induction by bacterially derived plasmid DNA does not contribute to the magnitude of the antibody response and is not required for the induction or short-term maintenance of DNA-induced CTL. However, IFNgamma is necessary for the development of IgG2a antibodies that may be crucial for protection against some pathogens.     

Improvement of atopic dermatitis in NC/Nga mice by topical application of CpG phosphodiester-ODN

Atopic dermatitis (AD) is a chronic inflammatory skin disease with pruritic and eczematous lesions characterized by increased total IgE level, inflammatory cell infiltration, and the elevated expression of Th2 cytokines. Synthetic oligodeoxynucleotides containing unmethylated CpG dinucleotides are known to have immunostimulatory activities in mice and to convert from Th2 to Th1 immune responses in AD. Previous work has shown clinical effectiveness of CpG phosphorothioate-ODN in AD mice model. However, due to longer in vivo half-life and the possibility of causing unwanted side effects, therapeutic use of CpG phosphorothioate-ODN can be limited. Thus, we investigated the efficacy of CpG phosphodiester-ODN with a novel sequence in NC/Nga mice. Topical application of phosphodiester-ODN penetrated rapidly from epidermis to the lymph nodes, accompanied by reduced infiltration of inflammatory cells and decreased number of cells expressing cytokines such as IL-4, IL-10 and IFN-gamma. Furthermore, the expression of IFN-gamma was reduced in the CpG ODNs-treated NC/Nga mice while the expression of IL-12p40 was increased, suggesting stimulation of Th1 immune response. The expression of IL-10 was strongly reduced, which meant the suppression of Th2 immune response in NC/Nga mice, accompanied by reduced level of IgE and IgG1, but increased level of IgG2a in sera. Since phosphodiester-ODN has been shown to cause minimum side effect comparing its phosphorothioate counterpart, it is proposed to become a new therapeutic modality for AD.     

MUC1基因疫苗诱导小鼠特异性CTL和体液免疫应答

目的 :观察MUC1基因疫苗诱导小鼠特异性杀伤性T细胞及体液免疫应答的作用。方法 :采用股四头肌肌肉注射 ,将构建的MUC1基因疫苗pcDNA3.1 MUC1免疫雌性BALB/c小鼠 ,每次间隔 3wk ,共 3次。最后 1次免疫后第 3周 ,接种表达MUC1的EMT6乳腺癌细胞进行免疫保护实验。用 4h51Cr释放法检测小鼠脾细胞特异性CTL杀伤活性 ;免疫组化染色法检测小鼠血清特异性抗体的水平。结果 :在效靶比为 10 0∶1、5 0∶1、2 5∶1、12 .5∶1时 ,MUC1基因疫苗免疫组特异性CTL对EMT6靶细胞杀伤活性分别为 5 4 .1%、39.8%、2 6 .4 %和2 0 .1% ,对照组分别为 13.2 %、10 .0 %、8.2 %、7.2 %和 11.7%、9.8%、7.7%、7.0 % ,前者与后二者差异显著 (P <0 .0 1)。免疫组化染色检测显示 ,人乳腺癌组织MUC1呈染色阳性 ;MUC1基因疫苗免疫组仅见 4 0 % (4/ 10 )的小鼠有肿瘤形成 ,而 pcDNA3.1对照组和生理盐水阴性对照组 10 0 %可见肿瘤形成、生长 ,表明MUC1基因疫苗免疫组小鼠具有一定的免疫保护作用。结论 :MUC1基因疫苗可诱导小鼠产生特异性CTL及体液免疫应答 ,对小鼠体内荷瘤可能具有一定的预防作用     

CD146基因重组质粒的构建及蛋白表达

目的构建CD146原核、真核表达载体,证实融合蛋白在原核细胞的诱导表达以及在胃癌细胞内的表达和定位。方法提取人黑色素瘤细胞A875的总mRNA并进行反转。以反转录的cDNA为模板PCR扩增CD146全长编码基因,分别克隆至pCDNA3.1-myc/his以及pGEX-4T-3表达载体中。原核重组质粒鉴定后转入BL21细胞中并经了诱导表达及纯化,真核表达质粒转入胃癌MKN45细胞中,分别利用westernblot和激光共焦扫描显微技术检测了重组质粒的表达以及在胃癌细胞中的定位。结果 CD146全长基因序列克隆到原核、真核表达载体中,酶切鉴定片段为1930bp。原核诱导出了GST-CD146并进行了纯化,CD146在真核细胞中表达为113KD的糖蛋白,Westernblot检测到真核转染的myc/his-CD146表达,条带约为120KD,免疫荧光显示蛋白定位在胃癌MKN细胞膜。结论成功构建了CD146原核、真核表达载体,融合蛋白在胃癌MKN45细胞表达。     

DNA免疫激发针对肾母细胞瘤CTL效应初步研究

目的 将鼠源肾母细胞瘤WT1基因一段序列(WT1y)构建于真核表达质粒pCDNA3.1( ),通过小鼠和体外细胞模型初步研究所激发的针对肾母细胞瘤CTL效应.方法 人工合成WT1基因一段序列,含有HLA-A*2402锚定残基的9个氨基酸.构建重组真核表达质粒pCDNA3.1( )/WT1y.免疫Balb/c小鼠,通过ELISA方法检测体液免疫反应;分离脾淋巴细胞,FCM检测脾淋巴细胞中CD4/CD8;将分离的脾淋巴细胞与小鼠肾母细胞瘤细胞共培养,检测其体外溶解组织相容性抗原型别一致的外源性靶细胞能力.结果 构建的重组质粒经测序鉴定,与GenBank中登录的序列完全一致;免疫组小鼠T淋巴细胞增殖及CTL活性均明显优于对照组(P＜0.01);体外具有较强溶解靶细胞的功能.结论 WT1基因的DNA疫苗初步研究具有很强的CTL效应,为小儿肾母细胞瘤的治疗提供了新的思路.