日本血吸虫22.6kDa抗原T细胞表位的重组、表达及初步鉴定

目的　鉴定日本血吸虫中国大陆株22.6kDa抗原(Sj22.6)的T细胞表位。　方法　用计算机软件预测Sj22.6分子的T细胞表位,设计并合成其编码核苷酸,定向克隆入融合表达载体pET32c(+),转化大肠杆菌BL21感受态细胞,经酶切及测序鉴定出重组克隆。阳性克隆经IPTG诱导表达,表达产物用NTA柱纯化。用纯化后的表位肽融合蛋白体外刺激C3H小鼠脾单个核细胞,3HTdR掺入法检测其增殖。　结果　用计算机软件预测获得Sj22.6抗原的4个候选表位肽,并成功克隆入pET32c(+)。其重组体均可表达分子量约20kDa的融合蛋白,用NTA柱纯化后经12%聚丙烯酰胺凝胶电泳(SDSPAGE)显示单一条带。其中P4、P5、P6融合蛋白能有效刺激小鼠脾单个核细胞增殖。　结论　从Sj22.6抗原中初步鉴定出P4、P5、P63个T细胞表位     

人胰腺癌Hedgehog信号通路中膜蛋白受体PTCH的纯化

目的 构建人胰腺癌PTCH基因表达载体并诱导融合蛋白表达。方法 从人胰腺癌细胞株SW1990抽提总RNA，经RT—PCR扩增出PTCH基因，经纯化、回收目的基因PTCH，将其插入表达载体pET22b，转化E．Coli BL21-CodonPlus^（TM）-RP，构建重组质粒pET22b／PTCH，IPTG诱导表达融合蛋白，免疫印迹进行鉴定，Ni-螯合亲和层析纯化融合蛋白。结果 从人胰腺癌细胞株SW1990克隆出长为789bp PTCH目的片段，成功构建重组质粒pET22b／PTCH，并诱导表达目的蛋白；经Ni-螯合亲和层析得到纯化的融合蛋白。结论 成功构建了重组质粒pET22b／PTCH，并获得纯化的融合蛋白，为进一步研究Hedgehog信号通路在胰腺癌中的发病机制提供了有效工具。     

重组人神经肽Y融合蛋白的表达、纯化及生物信息学分析研究

目的 在大肠杆菌中表达人神经肽Y,并对之进行纯化、鉴定及生物信息学分析.方法 取已构建好且经测序确认无误的重组质粒pET28a-NPY转化大肠杆菌BL21(DE3),IPTG诱导表达融合蛋白,并经SDS-PAGE检测和Western印迹鉴定,表达产物包涵体经Ni2+-NTA亲和层析纯化.然后利用相关在线软件进行生物信息学分析NPY蛋白.结果 经IPTG诱导含有pET28a-NPY重组质粒的DE3菌,表达出重组人NPY融合蛋白.重组蛋白经Ni2+-NTA亲和层析进行纯化后,得到了较高纯度的融合蛋白.经相关在线软件分析后获得了NPY的相关生物学特性.结论 重组质粒pET28a-NPY在大肠杆菌DE3中成功表达,亲和层析纯化后获得较高纯度融合蛋白,并对NPY蛋白的生物学特征进行了预测,为进一步研究其生物学功能及其抗体的研制奠定了基础.     

结核杆菌H37Rv株重组KatG蛋白的表达纯化研究

目的表达纯化结核杆菌H37Rv株重组KatG（简称rKatG）蛋白，为深入研究异烟肼耐药机制奠定基础。方法重组质粒pET23b—KatG转化大肠埃希菌表达菌株BL21（DE3）plysE,IPTG诱导rkatG蛋白表达。经SDS-PAGE电泳和Western blot鉴定后，优化表达条件，用镍离子鳌合亲和层析柱纯化rKatG蛋白，对纯化产物进行过氧化氢酶活性测定。结果成功构建了重组质粒pET、23b-KatG,rKatG蛋白以可溶性蛋白形式表达，经亲和层析后的rKatG蛋白纯度为95．6％，过氧化氢酶试验阳性。结论构建的pET23b-KatG重组质粒能高效表达有酶活性的可溶性rKatG蛋白，经亲和层析后可得到高纯度的纯化蛋白。     

人胰腺癌 Hedgehog 信号通路中膜蛋白受体 PTCH 的纯化

目的 构建人胰腺癌PTCH基因表达载体并诱导融合蛋白表达.方法 从人胰腺癌细胞株SW1990抽提总RNA,经RT-PCR扩增出PTCH基因,经纯化、回收目的 基因PTCH,将其插入表达载体pET22b,转化E. Coli BL21-CodonPlus(TM)-RP,构建重组质粒pET22b/PTCH,IPTG诱导表达融合蛋白,免疫印迹进行鉴定,Ni-螯合亲和层析纯化融合蛋白.结果 从人胰腺癌细胞株SW1990克隆出长为789 bp PTCH目的 片段,成功构建重组质粒pET22b/PTCH,并诱导表达目的 蛋白;经Ni-螯合亲和层析得到纯化的融合蛋白.结论 成功构建了重组质粒pET22b/PTCH,并获得纯化的融合蛋白,为进一步研究Hedgehog信号通路在胰腺癌中的发病机制提供了有效工具.     

粪肠球菌心内膜炎抗原efaA蛋白的原核表达及纯化

目的原核表达纯化粪肠球菌心内膜炎抗原efaA蛋白,为粪肠球菌心内膜炎的致病机制研究及临床血清学诊断奠定基础。方法从粪肠球菌中扩增efaA基因,相应酶切后,克隆到原核表达载体pET30a中,构建pET30a-efaA重组质粒。经BamhI、XhoI酶切及测序鉴定,将pET30a-efaA质粒转化入BL21(DE3)。以IPTG诱导BL21(DE3)表达efaA融合蛋白,亲和层析纯化重组蛋白,SDS-PAGE、WesternBlot分析鉴定。结果PCR体外扩增efaA基因产物约943bp,重组表达质粒pET30a-efaA在大肠杆菌BL21(DE3)中表达,通过亲和层析获得纯化重组蛋白。SDS-PAGE、Western免疫印迹显示蛋白表达带的分子量约为34kd。结论粪肠球菌心内膜炎抗原efaA蛋白在大肠杆菌BL21(DE3)中成功表达并纯化。     

重组人神经肽Y受体Y1融合蛋白的表达、纯化及其生物信息学分析研究

目的 在大肠杆菌中表达人神经肽Y Y1受体,并对之进行纯化、鉴定及生物信息学分析.方法 取已构建好且经测序确认无误的重组质粒pET28a-Y1转化大肠杆菌BL21(DE3),IPTG诱导表达融合蛋白,并经SDS-PAGE检测和Western Blot鉴定,表达产物包涵体经Ni2+-NTA亲和层析纯化.然后利用相关在线软件进行生物信息学分析Y1受体蛋白.结果 经IPTG诱导含有pET28a- Y1重组质粒的DE3菌,表达出重组人Y1融合蛋白.重组蛋白经Ni2+-NTA亲和层析进行纯化后,得到了较高纯度的融合蛋白.经相关在线软件分析后获得了Y1受体的相关生物学特性.结论 重组质粒pET28a-Y1在大肠杆菌DE3中成功表达,亲和层析纯化后获得较高纯度融合蛋白,并对Y1受体蛋白的生物学特征进行了预测,为进一步研究其生物学功能及其抗体的研制奠定了基础.     

重组人神经肽Y受体Y5融合蛋白的表达、纯化及其生物信息学分析研究

目的 在大肠杆菌中表达人神经肽Y Y5受体,并对之进行纯化、鉴定及生物信息学分析.方法 取已构建好且经测序确认无误的重组质粒pET28a-Y5转化大肠杆菌BL21(DE3),IPTG诱导表达融合蛋白,并经SDS-PAGE检测和Western印迹鉴定,表达产物包涵体经Ni2+-NTA亲和层析纯化.然后利用相关在线软件进行生物信息学分析Y5受体蛋白.结果 经IPTG诱导含有pET28a-Y5重组质粒的DE3菌,表达出重组人Y5融合蛋白.重组蛋白经Ni2+-NTA亲和层析进行纯化后,得到了较高纯度的融合蛋白.经相关在线软件分析后获得了Y5受体的相关生物学特性.结论 重组质粒pET28a-Y5在大肠杆菌DE3中成功表达,亲和层析纯化后获得较高纯度融合蛋白,并对Y5受体蛋白的生物学特征进行了预测,为进一步研究其生物学功能及其抗体的研制奠定了基础.     

重组人神经肽Y受体Y2融合蛋白的表达、纯化及其生物信息学分析研究

目的 在大肠杆菌中表达人神经肽Y Y2受体,并对之进行纯化、鉴定及生物信息学分析.方法 取已构建好且经测序确认无误的重组质粒pET28a-Y2转化大肠杆菌BL21(DE3),IPTG诱导表达融合蛋白,并经SDS-PAGE检测和Western 印迹鉴定,表达产物包涵体经Ni~(2+)-NTA亲和层析纯化.然后利用相关在线软件进行生物信息学分析Y2受体蛋白.结果 经IPTG诱导含有pET28a-Y2重组质粒的DE3菌,表达出重组人Y2融合蛋白.重组蛋白经Ni~(2+)-NTA亲和层析进行纯化后,得到了较高纯度的融合蛋白.经相关在线软件分析后获得了Y2受体的相关生物学特性.结论 重组质粒pET28a-Y2在大肠杆菌DE3中成功表达,亲和层析纯化后获得较高纯度融合蛋白,并对Y2受体蛋白的生物学特征进行了预测,为进一步研究其生物学功能及其抗体的研制奠定了基础.     

户尘螨Ⅱ类变应原Der p2 T细胞表位融合基因的克隆和原核表达

目的原核表达、纯化户尘螨(Dermatophagoides pteronyssinus)主要变应原Der p2的T细胞表位融合肽。方法将已报道的户尘螨Der p2中编码4个T细胞表位(T1~T4)的核苷酸序列以T1-T2-T3-T4的方式连接,人工合成为嵌合基因,命名为Der p2 T。PCR扩增目的基因Der p2 T,将纯化的扩增片段克隆至p ET-28a(+)载体,构建原核重组表达质粒p ET-28a(+)-Der p2 T,并进行双酶切验证。大量诱导表达含p ET-28a(+)-Der p2T的E.coli BL-21菌株,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,经Ni-NTA亲和层析获得纯化重组蛋白,并进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹(Western blotting)分析。ELISA法检测重组Der p2 T细胞表位融合肽对户尘螨过敏患者血清Ig E抗体的结合能力。结果双酶切结果表明,构建了原核重组表达质粒p ET-28a(+)-Der p2 T。SDS-PAGE分析结果显示,获得相对分子质量(Mr)为10 000的重组Der p2 T细胞表位融合肽。Western blotting分析结果显示,纯化了Der p2的T细胞表位融合肽。Der p2 T细胞表位融合肽对粉尘螨哮喘患者的血清Ig E结合能力[(37.70±9.89)μg/ml]较Der p2显著降低[(85.89±9.63)μg/ml](P0.01)。结论制备Der p2 T细胞表位融合肽。与Der p2相比,重组Der p2 T细胞表位融合肽对户尘螨过敏患者血清Ig E抗体的结合能力明显降低。     

A "public" T-helper epitope of the E7 transforming protein of human papillomavirus 16 provides cognate help for several E7 B-cell epitopes from cervical cancer-associated human papillomavirus genotypes.

We have identified a major T-cell epitope, amino acids 48-54 (DRAHYNI, in one-letter code) in the E7 open reading frame protein of human papillomavirus (HPV) type 16. Lymph node cells from mice immunized with synthetic peptides containing DRAHYNI proliferated and produced interleukin when challenged in vitro with peptide or whole HPV-16 E7 fusion protein. The T epitope was recognized in association with all five major histocompatibility complex class II I-A and I-E alleles tested. Synthetic peptides consisting of DRAHYNI linked to major B-cell epitopes on the E7 molecule formed immunogens capable of eliciting strong antibody responses to HPV-16 E7. The T epitope could provide help for the production of antibody to several B epitopes simultaneously, including a B epitope of HPV-18 E7 protein. Mice immunized with a peptide containing DRAHYNI and B epitope and, at a later date, infected with recombinant vaccinia E7 virus, displayed secondary antibody responses to E7. Because E7 has a role in cell transformation and is the most abundant viral protein in HPV-associated neoplastic cervical epithelial cells, the data have implications for vaccine strategies.     

The proteolytic fragments generated by vertebrate proteasomes: structural relationships to major histocompatibility complex class I binding peptides.

Proteasomes are involved in the proteolytic generation of major histocompatibility complex (MHC) class I epitopes but their exact role has not been elucidated. We used highly purified murine 20S proteasomes for digestion of synthetic 22-mer and 41/44-mer ovalbumin partial sequences encompassing either an immunodominant or a marginally immunogenic epitope. At various times, digests were analyzed by pool sequencing and by semiquantitative electrospray ionization mass spectrometry. Most dual cleavage fragments derived from 22-mer peptides were 7-10 amino acids long, with octa- and nonamers predominating. Digestion of 41/44-mer peptides initially revealed major cleavage sites spaced by two size ranges, 8 or 9 amino acids and 14 or 15 amino acids, followed by further degradation of the latter as well as of larger single cleavage fragments. The final size distribution was slightly broader than that of fragments derived from 22-mer peptides. The majority of peptide bonds were cleaved, albeit with vastly different efficiencies. This resulted in multiple overlapping proteolytic fragments including a limited number of abundant peptides. The immunodominant epitope was generated abundantly whereas only small amounts of the marginally immunogenic epitope were detected. The frequency distributions of amino acids flanking proteasomal cleavage sites are correlated to that reported for corresponding positions of MHC class I binding peptides. The results suggest that proteasomal degradation products may include fragments with structural properties similar to MHC class I binding peptides. Proteasomes may thus be involved in the final stages of proteolytic epitope generation, often without the need for downstream proteolytic events.     

C2株蓝氏贾第鞭毛虫组蛋白H3基因的克隆表达与基因分析

目的 克隆表达C2株蓝氏贾第鞭毛虫H3组蛋白基因,与WB株蓝氏贾第鞭毛虫进行同源分析,构建C2株蓝氏贾第鞭毛虫H3组蛋白分子进化树。方法 PCR扩增获取H3组蛋白基因,构建pGM-T-H3重组载体,转化E. coli TOP10感受态宿主细胞,挑选阳性克隆并进行序列分析,利用限制性内切酶NcoⅠ和 XhoⅠ构建pET28a(+)-H3重组载体,转化E. coli Rosetta( DE3),IPTG诱导组蛋白H3表达,Western blotting鉴定表达,与美国WB株蓝氏贾第鞭毛虫及模式生物H3组蛋白基因和蛋白序列进行同源分析。结果 成功克隆表达C2株蓝氏贾第鞭毛虫H3组蛋白基因,同源比对结果显示C2株蓝氏贾第鞭毛虫基因序列与美国WB株完全一致,但与其他现存真核生物亲缘关系较为疏远。结论 C2株蓝氏贾第鞭毛虫H3组蛋白分子进化树分析表明贾第虫H3组蛋白基因在进化过程中与其他物种分化较早,本研究结果为进一步研究蓝氏贾第鞭毛虫的生物进化地位提供有价值的实验资料。     

Preclinical development of an adjuvant-free peptide vaccine with activity against CMV pp65 in HLA transgenic mice

Epitope vaccines have shown promise for inducing cellular immune responses in animal models of infectious disease. In cases where cellular immunity was augmented, peptide vaccines composed of covalently linked minimal cytotoxic T-lymphocyte (CTL) and T-helper (T(H)) epitopes generally showed the most efficacy. To address a clinical vaccine strategy for cytomegalovirus (CMV) in the context of HCT (hematopoietic cell transplantation), we observed that linking the synthetically derived pan-DR epitope peptide (PADRE) or one of several tetanus T(H) epitopes to the immunodominant human leukocyte antigen (HLA) A*0201-restricted CTL epitope from CMV-pp65 to create a fusion peptide caused robust cytotoxic cellular immune responses in HLA A*0201/K(b) transgenic mice. Significantly, the fusion peptides are immunogenic when administered in saline solution by either subcutaneous or intranasal routes. CpG-containing single-stranded DNA (ss-oligodeoxynucleotide [ODN]) added to the fusion peptides dramatically up-regulated immune recognition by either route. Notably, target cells that either expressed full-length pp65 protein from vaccinia viruses or were sensitized with the CTL epitope encoded in the vaccine were recognized by splenic effectors from immunized animals. Visualization of murine peptide-specific CTL by flow cytometry was accomplished using an HLA A*0201 tetramer complexed with the pp65(495-503) CTL epitope. T(H)-CTL epitope fusion peptides in combination with CpG ss-ODN represent a new strategy for parenteral or mucosal delivery of vaccines in a safe and effective manner that has applicability for control or prophylaxis of infectious disease, especially in situations such as vaccination of donors or recipients of HCT, where highly inflammatory adjuvants are not desired.     

Heat-shock protein 90 associates with N-terminal extended peptides and is required for direct and indirect antigen presentation

CD8(+) T cells recognize peptide fragments of endogenously synthesized antigens of cancers or viruses, presented by MHC I molecules. Such antigen presentation requires the generation of peptides in the cytosol, their passage to the endoplasmic reticulum, loading of MHC I with peptides, and transport of MHC I-peptide complexes to the cell surface. Heat-shock protein (hsp) 90 is a cytosolic chaperone known to associate with peptide and peptide precursors of MHC I epitopes. We report here that treatment of cells with hsp90 inhibitors leads to generation of "empty" MHC I caused by inhibited loading of MHC I with peptides. Inhibition of hsp90 does not inhibit synthesis of MHC I, nor does it affect the activity of proteasomes. Hsp90-inhibited cells, such as proteasome-inhibited cells, are poor stimulators of T lymphocytes. The role of hsp90 in presentation of an ovalbumin epitope is shown to be at a postproteasomal step: hsp90 associates with N-terminally extended precursors of the SIINFEHL epitope, and such peptides are depleted from hsp90 preparations in hsp90-inhibited cells. Inhibition of hsp90 in the antigen donor cell compromises their ability to cross-prime. Conversely, stressed cells expressing elevated hsp90 levels show a heat-shock factor-dependent, enhanced ability to cross-prime. These results demonstrate a substantial role for hsp90 in chaperoning of antigenic peptides in direct and indirect presentation. The introduction of a stress-inducible component in these pathways has significant implications for their modulation during fever and infection.     

Cloning in Escherichia coli and physical structure of hepatitis B virion DNA.

A restriction map of hepatitis B virion DNA was established after cloning of the whole viral genome in Escherichia coli. By use of EcoRI, Xho I, Bgl II, Xba I, BamHI, HincII, and Hae III endonucleases, a total of 28 restriction sites were mapped. The single-stranded region was localized on the restriction map and 5' end of the short strand was mapped at a fixed position.     

Cloned restriction/modification system from Pseudomonas aeruginosa.

DNA fragments from Pseudomonas aeruginosa carrying the PaeR7 restriction/modification genes have been cloned in the plasmid vector pBR322 and propagated in Escherichia coli. A subclone (pPAORM3.8) has been constructed that contains the complete restriction/modification system on a 3.8-kilobase DNA fragment. Digestion of the pPAORM3.8 plasmid with nuclease BAL-31 has yielded two types of clones. One type contains an active methylase gene but no active endonuclease gene; such clones will modify the DNA but not restrict the growth of incoming phage in vivo. The second type contains an active endonuclease gene but no active methylase gene, as judged both by in vivo tests and by the activity of the cell extracts in vitro. Although extracts of cells containing these plasmids display restriction endonuclease activity, these bacteria are unable to restrict the growth of incoming phage. Furthermore, chromosomal and phage DNA isolated from these host cells are not protected against cleavage by PaeR7 in vitro. The properties of PaeR7 endonuclease and methylase enzymes have also been examined. The PaeR7 restriction endonuclease recognizes and cleaves the sequence C decreased T-C-G-A-G, as does Xho I. However, there exists a canonical Xho I site at 26.5% on the adenovirus 2 genome which is totally refractory to PaeR7 cleavage but is cut by Xho I. Under conditions of low salt, high glycerol, and high enzyme concentrations, a "PaeR7" activity is found that is similar to that observed for EcoRI. Finally, evidence is presented that the PaeR7 methylase modifies the adenine residue within the recognition sequence.     

弓形虫主要表面抗原P30的克隆、表达与纯化

目的通过分子克隆技术获取弓形虫主要表面抗原P30蛋白。方法自行设计引物,通过PCR扩增获得P30基因片段,采用EcoRⅠ、XhoⅠ双酶切,定向克隆到载体pThioHis中,转化大肠杆菌Top10,利用酶切、DNA序列分析鉴定阳性克隆,异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达,融合蛋白通过镍结合树脂(ProBond~(TM)Resin)进行纯化,并用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹(Westernblotting)鉴定。结果PCR、酶切、连接的产物经电泳鉴定,均与预期设计相符合。DNA序列分析结果表明,除一个同义突变,其余均与文献报道相符。IPTG诱导表达后经层析纯化获得46kDa含P30的融合蛋白。结论通过定向克隆、表达与纯化,获得含P30的融合蛋白。     

人端粒酶逆转录酶抗原HLA-A0201限制性CTL表位预测及鉴定

目的应用生物信息学方法对人端粒酶逆转录酶(h TERT)HLA-A2+限制性细胞毒性T细胞(CTL)表位进行预测和鉴定,寻找诱导机体特异性杀伤肺癌肿瘤细胞的抗原表位。方法应用生物信息学软件BIMAS、SYFPEITHI对h TERT蛋白进行HLA-A0201限制性CTL抗原表位预测,筛选优势表位;应用肽亲和力实验、乳酸脱氢酶(LDH)释放实验及人干扰素γ(IFN-γ)ELISPOT实验验证表位,筛选出激发机体产生特异性免疫反应的表位。结果生物信息学软件筛选出优势表位为:ILAKFLHWL、ELLRSFFYV及ILSTLLCSL;肽亲和力实验得到优势表位荧光系数(FI)为:ILAKFLHWL0.67、ELLRSFFYV0.66及ILSTLLCSL0.90;LDH释放实验显示ILAKFLHWL所诱导CTLs的杀伤率明显高于其它各表位,也明显高于阴性表位,差异均具有统计学意义(P0.05);人IFN-γELISPOT实验证明ILAKFLHWL所诱导的CTLs产生的IFN-γ斑点数多于其他表位,差异具有统计学意义(P0.05)。结论ILAKFLHWL的免疫原性强,可用于后续制备肺癌多肽疫苗。     

Impact of intrapeptide epitope location on CD8 T cell recognition: implications for design of overlapping peptide panels

BACKGROUND: Antigen-specific CD8 T cells following infection or immunization are typically assessed by measuring interferon-gamma production after stimulation with overlapping peptides spanning the region of interest. The effect of epitope location within such peptides is not known but may influence recognition. OBJECTIVE: To examine if peptides containing the appropriate C-terminal anchor amino acid residue would provide more sensitive detection of T cell responses. The impact was examined of epitope location within overlapping peptides on recognition of epitope-specific CD8 T cell responses. METHODS: C-terminal amino acid residues were analyzed in well-defined optimal epitopes for HIV, Epstein-Barr virus, cytomegalovirus and influenza and in peptide-binding motifs. Recognition of known epitopes within longer synthesized peptides by peripheral blood mononuclear cells or CD8 T cell lines was tested using interferon-gamma Elispot at various peptide concentrations. RESULTS: Only 9 of 20 amino acids served as the C-terminal anchor position in 96% of described optimal epitopes and in 95% of peptide-binding motifs. A CD8 T cell response to an epitope within a longer peptide is best detected when the epitope is situated at the C-terminal end of the longer peptide, both when using peptides designed to include the optimal epitope at every possible position and when comparing responses towards optimal epitopes and corresponding overlapping peptides in a larger group of subjects. CONCLUSION: When using overlapping peptides to screen for CD8 T cell responses, more sensitive detection will be achieved using known C-terminal anchor amino acid residues at the C-terminus.