本文選題：B細(xì)胞淋巴瘤　切入點(diǎn)：融合型獨(dú)特型DNA疫苗　出處：《河北醫(yī)科大學(xué)》2008年碩士論文

【摘要】： 目的 B細(xì)胞淋巴瘤的膜表面免疫球蛋白(surface membrane immunoglobulin,mIg)獨(dú)特型(idiotype,Id)來自基因重排,由可變區(qū)的重鏈(VH)和輕鏈(VL)基因所決定,其突出的個(gè)體化特征即腫瘤特異性抗原(TSA)正好滿足了抗腫瘤疫苗的特異性的需要。將來源于個(gè)體的惡性增殖的B細(xì)胞的Ig特異性片段VH和VL通過基因克隆制備成單鏈抗體可變區(qū)片段(scFv),二者由一段短肽連接,可以模擬完整免疫球蛋白的免疫原性。由于scFv源于自身抗原,單獨(dú)應(yīng)用不足以誘導(dǎo)抑制腫瘤細(xì)胞的免疫反應(yīng),為此人們?cè)谔岣呙庖咴苑矫孀隽舜罅康难芯俊Ｑ芯空甙l(fā)現(xiàn)利用配體與細(xì)胞表面受體的結(jié)合可提高抗原呈遞給抗原提呈細(xì)胞(APC)的效率,提高抗原特異性免疫反應(yīng)。Biragyn將單核細(xì)胞趨化蛋白3(Monocyte chemotactic protein-3,MCP3)與scFv偶聯(lián)作為疫苗免疫小鼠,發(fā)現(xiàn)其表達(dá)的融合蛋白具有Ig的天然構(gòu)象及趨化因子的功能,能和抗原結(jié)合,同時(shí)吸引抗原提呈細(xì)胞(特別是未成熟樹突狀細(xì)胞)向抗原靠近,誘導(dǎo)的免疫反應(yīng)強(qiáng)于免疫球蛋白疫苗。同時(shí),抗腫瘤的DNA疫苗也是最近研究的一個(gè)熱點(diǎn),本研究利用小鼠B細(xì)胞淋巴瘤細(xì)胞株A20的scFv和MCP3的融合基因構(gòu)建DNA疫苗,并用該疫苗免疫同源小鼠,LDH法檢測(cè)小鼠的脾細(xì)胞與靶細(xì)胞A20孵育后的CTL活性。比較表達(dá)scFv和MCP3-scFv的DNA疫苗產(chǎn)生的細(xì)胞免疫反應(yīng)對(duì)腫瘤細(xì)胞的殺傷效果,探討融合型獨(dú)特型DNA疫苗在抗腫瘤免疫反應(yīng)中是否能誘導(dǎo)T細(xì)胞介導(dǎo)的抗腫瘤反應(yīng),誘導(dǎo)的抑瘤效果是否高于獨(dú)特型DNA疫苗,為提高獨(dú)特型疫苗的免疫原性提供理論基礎(chǔ),為淋巴瘤疫苗的制備及臨床試驗(yàn)研究拓寬思路。 方法: 1真核表達(dá)質(zhì)粒的構(gòu)建 1.1 PCR引物設(shè)計(jì) 根據(jù)A20細(xì)胞IgVH cDNA、IgVL cDNA與MCP3序列分別設(shè)計(jì)MCP3-scFv和scFv的上下游引物,此部分實(shí)驗(yàn)已經(jīng)完成[1]。根據(jù)EGFP序列設(shè)計(jì)EGFP上下游引物,序列均來源于Genebank。 1.2 PCR擴(kuò)增目的片段 以pGLo/MCP3-scFv為模板,分別用MCP3上游引物和VL下游引物擴(kuò)增MCP3-scFv,VH上游引物和VL下游引物擴(kuò)增scFv。以含有EGFP基因序列的質(zhì)粒PCX-EGFP為模板,分別用引物EGFP上游引物和EGFP下游引物擴(kuò)增EGFP基因。 1.3 pGEM T-easy載體克隆MCP3-scFv、scFv、EGFP片段以及鑒定 1.4 DNA質(zhì)粒構(gòu)建將PCR產(chǎn)物MCP3-scFv、scFv、EGFP酶切后,定向克隆到真核表達(dá)載體pTARGET ,得到真核表達(dá)質(zhì)粒pTARGET/EGFP、pTARGET/scFv-EGFP和pTARGET/MCP3-scFv-EGFP,將三種質(zhì)粒轉(zhuǎn)化感受態(tài)細(xì)菌及酶切鑒定,選取陽性克隆測(cè)序。 2質(zhì)粒轉(zhuǎn)染及鑒定 將構(gòu)建的質(zhì)粒分別電穿孔法轉(zhuǎn)染至培養(yǎng)至對(duì)數(shù)生長(zhǎng)期的人臍靜脈內(nèi)皮細(xì)胞ECV-304,轉(zhuǎn)染后48小時(shí),在倒置熒光顯微鏡下觀察融合蛋白的表達(dá)。 3質(zhì)粒準(zhǔn)備和免疫小鼠 3.1質(zhì)粒純化 應(yīng)用QIAGEN/Endofree Plasmid Maxi,Giga Kit分離純化質(zhì)粒pTARGET/EGFP、pTARGET/scFv-EGFP、pTARGET/MCP3-scFv-EGFP。每種質(zhì)粒內(nèi)毒素水平控制在0.1 EU/ug以下,制備成為符合動(dòng)物實(shí)驗(yàn)要求的質(zhì)粒。 3.2 DNA疫苗免疫動(dòng)物 將18只Babl/c小鼠按隨機(jī)區(qū)組方法分成三組:pTARGET/EGFP組; pTARGET/scFv-EGFP組;pTARGET/MCP3-scFv-EGFP組。6只/組。在小鼠雙側(cè)股四頭肌肌內(nèi)注射2.5g/L鹽酸布比卡因,每側(cè)50μl。5天后每組質(zhì)粒取50微克溶至50微升0.9%NaCL分別注射于小鼠兩側(cè)的股四頭肌,接種三次,每次間隔兩周。 4 LDH釋放法測(cè)定CTL的特異性殺傷率于小鼠免疫結(jié)束后第5天取脾細(xì)胞作為效應(yīng)細(xì)胞,A20細(xì)胞為靶細(xì)胞,將效靶比例設(shè)置為20/1、50/1、100/1,應(yīng)用LDH釋放法測(cè)定CTL活性。 5統(tǒng)計(jì)學(xué)分析 CTL的殺傷率數(shù)據(jù)分析采用SPSS(13.0)統(tǒng)計(jì)軟件進(jìn)行,3組間比較采用方差分析,兩兩比較采用SNK-q檢驗(yàn),P0.05認(rèn)為有統(tǒng)計(jì)學(xué)意義。 結(jié)果: 1真核表達(dá)質(zhì)粒經(jīng)相應(yīng)的限制性內(nèi)切酶酶切鑒定均正確。測(cè)序結(jié)果顯示目的片段基因序列與相關(guān)文獻(xiàn)報(bào)道基本符合。 2細(xì)胞轉(zhuǎn)染及熒光觀察:融合基因真核表達(dá)質(zhì)粒轉(zhuǎn)染ECV后經(jīng)倒置熒光顯微鏡下觀察發(fā)現(xiàn),pTARGET/EGFP、pTARGET/scFv-EGFP和pTARGET/MCP3-scFv-EGFP在轉(zhuǎn)染后48h和72h均表達(dá)帶較強(qiáng)綠色熒光的蛋白,間接提示融合蛋白成功表達(dá)。 3各組CTL殺傷率(%)為:效靶比例為100/1時(shí):pTARGET/MCP3-scFv-EGFP組:68.45±5.72 ; pTARGET/scFv-EGFP組:14.20±4.39 ; pTARGET /EGFP組: 10.76±1.02。pTARGET/MCP3-scFv-EGFP組與其它兩組比較均有統(tǒng)計(jì)學(xué)差異(P0.05)。 結(jié)論: 1成功構(gòu)建兩種DNA疫苗: pTARGET/scFv-EGFP和pTARGET/MCP3-scFv-EGFP。 2 DNA疫苗免疫動(dòng)物后可誘導(dǎo)出特異性細(xì)胞免疫反應(yīng)。同時(shí)試驗(yàn)證明,與scFv疫苗相比,MCP3與scFv融合的獨(dú)特型DNA疫苗能誘導(dǎo)高效的抗腫瘤免疫應(yīng)答。
[Abstract]:objective
Surface membrane immunoglobulin B cell lymphoma (surface membrane, immunoglobulin, mIg) (idiotype, Id) unique type from gene rearrangement by the heavy chain variable region (VH) and light chain (VL) determined by individual genes, its prominent characteristic is tumor specific antigen (TSA) just to meet resistance tumor vaccine specific needs. The future source of malignant proliferation in individual B cells Ig specific fragment of VH and VL were prepared by gene clone fragment scFv antibody variable region (scFv), the two are connected by a short peptide, can simulate the complete immunoglobulin scFv due to immunogenicity. Due to the self antigen immune response alone is not sufficient to induce inhibition of tumor cells, so people in to do a lot of research to improve immunogenicity. The researchers found that using a combination of ligands and cell surface receptors can improve the antigens to antigen presenting cells (APC). To improve the efficiency of antigen-specific immune responses to.Biragyn monocyte chemotactic protein 3 (Monocyte chemotactic, protein-3, MCP3) and scFv coupling as vaccine in mice, we found that the expression of the fusion protein has the natural conformation of Ig and chemokines, and antigen binding, while attracting antigen-presenting cells (especially immature dendritic cells antigen) to close, the induced immune response in immunoglobulin vaccine. At the same time, a hot spot of anti-tumor DNA vaccine is a recent research, construct the fusion of mouse B cell lymphoma cell lines A20 scFv and MCP3 gene DNA vaccine used in this study, and the vaccine congenic mice, CTL the activity of mice were detected by LDH spleen cells and target cells incubated with A20. Expression of the lethal effect of cellular immune response to scFv and MCP3-scFv DNA vaccine on tumor cells, to investigate the fusion type Type DNA vaccine is unique in antitumor immunity can induce T cell mediated antitumor response induced by antitumor effect is higher than that of idiotypic DNA vaccine, providing theoretical basis for improving the immunogenicity of idiotype vaccine, for lymphoma vaccine preparation and clinical trial research broaden thinking.
Method:
Construction of 1 eukaryotic expression plasmids
1.1 PCR primer design
According to A20 cell IgVH cDNA, IgVL cDNA and MCP3 sequences, MCP3-scFv and scFv upstream and downstream primers were designed respectively. This part of experiments have been completed. [1]. is designed according to EGFP sequence. Upstream and downstream primers are derived from the EGFP.
1.2 PCR amplification of target fragments
Using pGLo/MCP3-scFv as template, MCP3 upstream primers and VL downstream primers were used to amplify MCP3-scFv. VH upstream primers and VL primers were used to amplify scFv., EGFP PCX-EGFP was used as template, and primer EGFP upstream primers and downstream primers were used to amplify the gene.
1.3 pGEM T-easy vector cloned MCP3-scFv, scFv, EGFP fragment and identification
1.4 DNA plasmid PCR products MCP3-scFv, scFv, EGFP after enzyme digestion and cloned into the eukaryotic expression vector pTARGET and eukaryotic expression plasmid pTARGET/EGFP, pTARGET/scFv-EGFP and pTARGET/MCP3-scFv-EGFP, three kinds of plasmids were transformed into competent bacteria and enzyme digestion, selected positive clones were sequenced.
Transfection and identification of 2 plasmids
The plasmids were transfected into human umbilical vein endothelial cells (ECV-304) cultured in logarithmic growth phase by electroporation respectively. After 48 hours of transfection, the expression of fusion protein was observed under inverted fluorescence microscope.
3 plasmids preparation and immunization of mice
3.1 plasmids purification
QIAGEN/Endofree Plasmid Maxi and Giga Kit were used to separate and purify plasmid pTARGET/EGFP, pTARGET/scFv-EGFP and pTARGET/MCP3-scFv-EGFP.. Each plasmid's level of endotoxin was controlled below 0.1 EU/ug, and the plasmid was prepared to meet the requirements of animal experiment.
3.2 DNA vaccine for immune animals
18 Babl/c mice were randomly divided into three groups: group pTARGET/EGFP group; pTARGET/scFv-EGFP group; pTARGET/MCP3-scFv-EGFP group.6 / group. Mice in bilateral femoral head four intramuscular injection of 2.5g/L bupivacaine hydrochloride, femoral head four muscle on each side of 50 l.5 days after each take 50 micrograms of plasmid solution were injected into the 0.9%NaCL to 50 L mice on both sides of the three dose, the time interval of two weeks.
The specific killing rate of CTL was determined by 4 LDH release method. Spleen cells were taken as effective cells fifth days after the end of immunization in mice, A20 cells were target cells, and the target target proportion was set to 20/1,50/1100/1. CTL activity was detected by LDH release method.
5 statistical analysis
The killing rate data of CTL were analyzed by SPSS (13) statistical software. The 3 groups were compared by ANOVA, and 22 compared with SNK-q test. P0.05 thought there was statistical significance.
Result:
1 eukaryotic expression plasmids were correctly identified by restriction endonuclease digestion. The sequencing results showed that the sequence of the target fragment was basically consistent with the related literature.
2 cell transfection and fluorescence observation: the fusion gene eukaryotic expression plasmid was transfected into ECV by inverted fluorescence microscope observation, pTARGET/EGFP, with a strong green fluorescence protein expression of pTARGET/scFv-EGFP and pTARGET/MCP3-scFv-EGFP in 48h and 72h after transfection, suggesting that indirect fusion protein was successfully expressed.
3, the killing rate (%) of CTL in each group was: the effective target ratio was 100/1: pTARGET/MCP3-scFv-EGFP group: 68.45 + 5.72; pTARGET/scFv-EGFP group: 14.20 + 4.39; pTARGET /EGFP group: 10.76 + 1.02.pTARGET/MCP3-scFv-EGFP group compared with other two groups (P0.05).
Conclusion:
1 successfully constructed two kinds of DNA vaccines: pTARGET/scFv-EGFP and pTARGET/MCP3-scFv-EGFP.
2 DNA vaccine can induce specific cellular immune response after animal immunization. Meanwhile, the test proved that compared with scFv vaccine, the idiotypic DNA vaccine fused with MCP3 and scFv can induce an effective anti-tumor immune response.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392;R733.1

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