【作者】 赵冰；

【导师】 潘崚； 王福旭；

【作者基本信息】 河北医科大学 ， 内科学， 2007， 硕士

【摘要】 目的:B细胞淋巴瘤细胞表面分泌的膜免疫球蛋白(smIg)是一种明确的肿瘤相关抗原(TAA),结合免疫佐剂主动免疫接种后可以激活体内免疫系统,靠自身的力量消灭体内残存的肿瘤细胞,为彻底治愈淋巴瘤提供可能。利用基因工程技术,将膜免疫球蛋白重链可变区基因(VH)和轻链可变区基因(VL)连接的单链小分子即scFv,可以模拟完整smIg的抗原性,但免疫原性弱,结合合适的免疫佐剂后,能增强其免疫原性,可以作为瘤苗进行免疫接种。本研究以BALB/c小鼠源B细胞性淋巴瘤细胞株A20为模型,制备smIg的scFv片段与免疫佐剂趋化因子MCP3融合的独特型蛋白疫苗,为进一步进行小鼠体内活性试验奠定基础。方法:重叠延伸拼接PCR技术,通过设计嵌合引物对两个不同目的序列进行连接。实验中,先后两次应用重叠延伸拼接PCR技术,制备小鼠B细胞性淋巴瘤细胞表面的独特型膜免疫球蛋白的scFv基因片段及scFv和MCP3融合基因片段。按重叠延伸PCR反应原理,设计6对引物,将Ig VH和Ig VL cDNA用一段编码(Gly4Ser)3连接肽的基因序列连接,然后,将scFv片段与免疫佐剂MCP3基因序列通过一段编码NDAQAPKS的spacer序列连接起来。其中,spacer序列可以保证scFv和MCP3蛋白正确折叠并保持各自独立的生物学活性。获得的scFv-MCP3融合基因片段经SacⅠ、KpnⅠ双酶切、定向克隆到原核表达质粒pGLo,再次酶切鉴定及测定核酸序列准确无误后转化到大肠杆菌中表达融合蛋白。1培养A20细胞,提取细胞内总RNA:B细胞性淋巴瘤细胞株A20培养于高糖DMEM培养基中,收获对数生长期的细胞,用Trizol试剂盒提取细胞内总RNA,琼脂糖电泳鉴定RNA的质量,分光光度计定量。2 RT-PCR法扩增IgVH、IgVL cDNA片段:以抽提的A20细胞内的总RNA为模板,六核随机引物法进行反转录。再以反转录产物为模板,分别以VH05’和VH0 3’、VL0 5’和VL0 3’为引物,扩增IgVH、IgVL cDNA片段。扩增产物电泳鉴定后进行核酸测序。3重叠延伸PCR法制备scFv基因片段:反应分两步进行:第一步,以IgVH、IgVL cDNA片段为模板,分别用特异性引物VH 5’和VH 3’、VL 5’和VL 3’对IgVH、IgVL cDNA片段进行修饰扩增,使IgVH cDNA下游末端与IgVLcDNA上游起始端出现互补结合区;第二步,等摩尔数的IgVH、IgVL cDNA修饰扩增片段为模板,加入同一反应体系中,变性后双链DNA解链,发生退火时,少量的互补末端的模板互补结合,互为模板并提供羟基末端,反向延伸,制备scFv片段。数个循环后加入引物VH 5’和VL 3’,按相同的PCR参数循环,得到一定量的IgVH cDNA和IgVL cDNA连接片段即scFv片段。4 MCP3基因的扩增:以含有MCP3基因序列的质粒为模板,用设计引物MCP3 5’和MCP3 3’扩增MCP3基因。5重叠延伸PCR法制备scFv与MCP3的融合基因片段:与制备scFv片段过程第二步相同:以等摩尔数的scFv基因片段和MCP3修饰片段为模板,以VH 5’和MCP3 3’为引物,连接制备scFv-MCP3融合基因片段。6 scFv-MCP3融合基因片段克隆质粒的构建:scFv-MCP3融合基因片段与pGEM-T载体连接,操作按说明书,用连接产物转化感受态细菌DH5α,涂到LB/氨苄/IPTG/X-Gal平板上,37°C培养过夜,挑取白色克隆,放大培养,提取质粒,进行PCR及SacⅠ、KpnⅠ双酶切鉴定阳性克隆,送大连宝生物公司测序。7 scFv-MCP3融合基因片段原核表达质粒的构建:经测序证实的阳性重组质粒经SacⅠ、KpnⅠ双酶切后,回收目的基因片段,然后经T4连接酶与同样处理的表达质粒载体pGLo进行连接,转化感受态细菌DH5α,涂布于LB/氨苄平板,37°C培养过夜,挑选数个菌落放大培养,抽提质粒。酶切及PCR鉴定阳性克隆。8 scFv-MCP3融合蛋白疫苗的表达:含有重组原核表达质粒的单克隆DH5α菌培养于LB培养基中,当菌液A600nm = 0.6左右时加入0.01%阿拉伯糖(L-arabinose)30°C低温诱导4小时,诱导前后菌体制样,10% SDS-PAGE凝胶电泳检测。结果:1基因片段的扩增及测序鉴定:RT-PCR扩增IgVH、IgVL cDNA的产物与预期大小一致,约400bp左右。测序结果显示IgVH、IgVL cDNA序列与文献报道一致,IgVH、IgVL基因序列翻译成氨基酸序列后与文献报道序列基本一致。测序后的IgVH、IgVL cDNA经相应的引物修饰后长度分别为407bp、401bp。以含MCP3基因的质粒为模板扩增MCP3基因,产物大小为335bp。经重叠延伸PCR获得scFv片段,产物为793bp,扩增scFv-MCP3融合基因片段,产物大小1111bp,与pGEM载体连接后挑选阳性的克隆测序,结果表明scFv和MCP3连接正确,并且无移码突变发生。在融合基因的制备扩增过程中,在IgVL序列内第231位核苷酸发生A→G碱基突变,其所在的读码框由ACA变为ACG,但均编码Thr,为无义突变,不影响蛋白表达后的氨基酸序列,未予修正。2重组表达质粒的酶切鉴定:重组表达质粒pGLo / scFv- MCP3被SacⅠ和KpnⅠ双酶切为约1100bp和5400bp两条片段。酶切结果表明成功构建表达质粒pGLo/scFv- MCP3。3融合蛋白质的表达:含重组表达质粒pGLo/scFv-MCP3的大肠杆菌DH5α培养于LB培养基中,阿拉伯糖30℃诱导4小时后可高效表达融合蛋白GFP-scFv-MCP3,SDS-PAGE电泳分析证实融合蛋白的表达量占菌体蛋白30%,融合蛋白的大小为65 kD。与预期结果相符。结论:1成功构建鼠源scFv片段与趋化因子MCP3融合的独特型B细胞淋巴瘤疫苗表达质粒pGLo / scFv- MCP3,并实现融合蛋白的初步表达。为融合蛋白疫苗体内活性检测准备实验基础。2重叠延伸拼接PCR过程中碱基与模板的错配率要明显高于常规PCR反应,即使使用保真性较好的聚合酶也不能避免错配的发生,可能导致碱基序列的点突变。测序结果验证序列拼接准确无误并且无移码突变后进行下一步实验。因此一定要使用保真性更高的DNA聚合酶,尽量避免点突变的发生。3可以对经典重叠延伸PCR技术的步骤进行简化,将等摩尔数的模板、引物同时加入反应体系中进行PCR扩增,就可以得到一定丰度的特异性扩增产物。4构建的表达质粒pGLo / scFv- MCP3中,MCP3的起始处和下游设计了BamH I的酶切位点,可通过酶切的方法将MCP3切出,并换入新的佐剂,进行免疫原性增强比较实验,方便了实验研究。更多还原

【Abstract】 Objective:The surface idiotype of immunoglobulin (Ig) expressed on B cell lymphoma cells is a kind of well-known tumor-associated antigen. Injectig the idiotype of Ig combined with a suitable adjuvant to a B cell lymphoma patient can activate patient’s immune system and, therefore, eliminate the minimal residual tumor cells by the activated immune system, which may be a novel and practical way to cure lymphoma. The DNA sequence of the single chain variable fragment (scFv) of Ig light chain can be combined with that of Ig heavy chain by gene recombination technology. After being transformed into special cells, the recombined gene can encode and transcriptate a“target”protein, which mimics the antigenicity of integrated Ig. But this“target”protein failed to elicit anti-tumor activity because the weakness of its immunogenicity.The present study is thus designed to clone the scFv gene of the idiotypic Ig from A20, a lymphoma cell line derived from the BABL/c mouse suffering from B cell malignancy, and then to recombine the scFv gene with the gene of monocyte chemotantic protein-3 (MCP3), a suitable adjuvant, and finally, to express the fusion protein vaccine in prokaryotic expressing system for further investigating the effect of idiotype fusion protein vaccine in vivo in animals.Methods:We have used the technique of splicing overlap extension by the polymerase chain reaction to hybrid two different genes together. In this study, we use Splicing by overlap extension by PCR (also recombinate PCR) technique for two times. Firstly, we cloned the cDNAs of immunoglobulin Ig VH and IgVL by RT-PCR and assembled them into the single-chain variable fragment(scFv) by recombinant PCR method. The cDNAs of IgVH and IgVL were connected by a gene fragment encoding a (Gly4Ser) 3 linker. Then, the fragments of scFv and MCP3 were connected with a gene sequence encoding a NDAQAPKS spacer, using recombinant PCR method again. The spacer will ensure the scFv and MCP3 protein folding respectively to maintain their natural function. The fusion gene of scFv-MCP3 were constructed correctly and cloned into the prokaryotic expression plasmid successfully identified by sequencing and restriction endonucleases SacⅠand KpnⅠ. The prokaryotic expression plasmid was transferred to E.coli DH5αto express fusion protein under induction.1 Cells culture and total RNA preparation: A20 cells derived from mouse B cell lymphoma were cultured in DMEM medium with high glucose. Cells were harvested at logarithmic growing phase. Total RNA was extracted using TRIzol Reagent according to the manufacturer’s instructions and the integrity of extracted RNA was confirmed by agarose electrophoresis.The amount of RNA was measured by spectrometry.2 Amplification of IgVH、IgVL cDNA fragments by reverse transcription PCR: The total RNA extracted from A20 cells was used as template. Random primers with six nucleotides and the reverse transcriptase of M-MLV were used for reverse transcription. The IgVH and IgVL cDNA fragments were specifically synthesized with primers VH 05’and VH0 3’, VL0 5’and VL0 3’, respectively. The PCR products were identified by electrophoresis on agarose gel and confirmed by DNA sequencing.3 Amplification of scFv fragment by overlap extension PCR: There are two steps. Firstly, the IgVH and IgVL cDNA fragments were modified by means of PCR using the specific primers VH 5’and VH 3’,VL 5’and VL 3’, respectively. The ends of the PCR products contain complementary sequences. Secondly, the two modified PCR products were mixed with equal quantity, denatured and reannealed; the single-stranded DNA strands having the complementary sequences annealed and then act as primers for each other. Amplified with primer VH 5’and VL 3’for several circles, the original IgVH and IgVL cDNA were spliced together into scFv.4 Amplification of MCP3 gene: The plasmid encoding MCP3 gene was used as template, primer MCP3 5’and MCP3 3’were applied to amplify MCP3 gene.5 Amplification of scFv-MCP3 fusion fragment by overlap extension PCR: The method of overlap extension PCR was the same as the construction of scFv. Templates of scFv and MCP3 fragments were mixed in equal quantity and primers of VH 5’and MCP3 3’were used to fuse scFv and MCP3 fragment.6 Construction of cloning plasmid encoding scFv-MCP3 fusion gene: scFv-MCP3 fusion gene and T cloning vector were ligated together according to the manufacturer’s instructions. The ligatition products were transformed to compentence E.coli DH5 cells. The transformed E.coli DH5 cells were planted onto LB plate,which had been duplicated with antibiotic,X-Gal. The cells were incubated overnight at 37°C. White clonies, which may be the inserts containing colonies, were picked up and macrocultureed. Plasmid DNA was extracted and screened by PCR, restriction endonucleases examination and DNA sequencing with primer T7 and Sp6.7 Construction of protocaryon expressing plasmid encoding scFv-MCP3 fusion gene: The correct clonal plasmid encoding scFv-MCP3 fusion gene and the expressing plasmid pGLo were both cleavaged by restriction endonucleases SacⅠand KpnⅠand the target genes were connected together with T4 ligase. The ligatition products were transformed to compentence E.coli DH5 cells. The transformed E.coli DH5 cells were planted onto LB plate, which had been duplicated with antibiotic. The cells were incubated overnight at 37°C. Several white clonies were picked up and macrocultured. Plasmid DNA was extracted, screened by PCR and restriction endonucleases examination. 8 Expression of scFv-MCP3 fusion protein: E.coli DH5αcells containing recombinant protocaryon expressing plasmid were grown in LB broth at 37°C. When OD600 of the culture became 0.6, L-arabinose was added at a final concentration of 0.01%. The culture was shaked at 30°C for 3.5 h to express target protein. Samples were collected from the culture and the target protein was detected on 10% SDS-PAGE.Results: 1 Amplification of target genes and their identifications by sequencing: The expected size of IgVH and IgVL cDNA fragments amplified by RT-PCR was about 400bp. The sequencing maps indicated: The nucleotide sequence of IgVH cDNA was the same as the data from Genebank. The size of the IgVH, IgVL cDNA and MCP3 modified fragment amplified by overlap extension PCR are 407bp, 401bp and 335bp. The scFv and scFv-MCP3 fragments splicinged by overlap extension were 793bp and 1111bp.The results of sequencing the recombinant cloning plasmid by primer T7 and Sp6 suggested that the scFv-MCP3 fusion gene were connected correctly without frameshift mutation. But there was a silent point mutation happened on the site of 231th nucleotide with A to G changing but the amino acids sequence was not changed. 2 Identification of the recombinant expression plasmid:The recombinant expression plasmid was digested by restriction endonucleases of SacⅠand KpnⅠinto two fragments, 5400bp and 1111bps, respectively. The result suggested that the expression plasmid pGLo/scFv-MCP3 was constructed successfully. 3 Expression of fusion protein: High yield of the fusion protein was expressed. And the fusion protein was 65 kD and account for 30% of the total protein of the bacteria analysised by SDS-PAGE.Conclusion:1 A prokaryotic plasmid, encoding the fusion gene of single-chain variable fragment with MCP3 and expressing idiotypic protein vaccination against B cell lymphoma, was constructed correctly and primarily expressed in DH5α. 2 The mismatching rate of gene splicing by overlap extension PCR is higher than common PCR. So it is necessary to use DNA polymerase of high fidelity to avoid point mutation. 3 We simplified the classic method of gene splicing by overlap extension PCR. Adding the equal-molar ratios’templates to the reaction system at the same time, the sufficient-specific products will be amplified after 30 cycles. 4 BamH I was designed on the origination and termination of MCP3 fragment in the recombinant expression plasmid pGLo/scFv-MCP3. It is convienent to cut MCP3 out to substitution other immune adjuvant.更多还原