草原兔尾鼠卵透明带3基因重组腺病毒载体的构建及表达

目的:探索制备免疫不育疫苗的新途径,获得草原兔尾鼠卵透明带3(LZP3)重组腺病毒减毒活疫苗。方法:将LZP3基因亚克隆到穿梭质粒pShuttle-CMV上,采用细菌内同源重组"两步转化法"构建携带LZP3基因的重组腺病毒载体。获得的重组腺病毒载体线性化后转染HEK293细胞,包装成病毒颗粒。PCR鉴定重组腺病毒,继而用鉴定正确的重组腺病毒感染HeLa细胞,并以RT-PCR、Western blot检测LZP3的转录和表达。结果:成功构建了携带LZP3基因的重组腺病毒pAd-LZP3载体,其转染293细胞后包装出重组病毒,PCR证实LZP3基因已整合至腺病毒基因组中,病毒的滴度可达1.2×1010pfu/L。RT-PCR和Western blot检测表明RAd-LZP3感染的HeLa细胞中LZP3基因能够有效转录和表达。结论:制备的RAd-LZP3可成功表达LZP3基因,为后续开展RAd-LZP3免疫动物的研究奠定了基础。     

草原兔尾鼠卵透明带3DNA疫苗pVAX1-sig-LTB-IZP3-C3d3的构建表达及其免疫不育的研究

目的:构建草原兔尾鼠卵透明带3 DNA疫苗pVAX1-sig-LTB-IZP3-C3d3进行小鼠的黏膜免疫,增强该疫苗的免疫不育效果.方法:将两种佐剂分子大肠杆菌不耐热肠毒素B亚基和C3d基因通过酶切鉴定,构建重组质粒pVAX1-sig-LTB-IZP3-C3 d3,采用RT-PCR和Western blot技术,检测其在mRNA和蛋白水平的表达,并通过肌肉注射、滴鼻和灌服3种途径免疫雌性C57BL/6小鼠,通过ELISA检测抗体水平及分型.结果:酶切鉴定,RT-PCR和Western blot 结果表明重组质粒构建正确并可在mRNA和蛋白水平的表达,ELISA结果表明以重组质粒pVAX1-sig-LTB-IZP3-C3d3免疫诱导的特异性IgG、IgA的水平明显高于对照组(P＜0.01)o抗生育实验表明该疫苗免疫的小鼠平均生仔数与对照组相比差异极显著(P＜0.01).结论:构建的草原兔尾鼠卵透明带3DNA疫苗重组质粒pVAX1-sig-LTB-IZP3-C3d3可高效地激发小鼠特异性免疫应答,提高抗生育的效果.     

草原兔尾鼠卵透明带3DNA疫苗pVAX1-sig-LTB-lZP3-C3d3的构建表达及其免疫不育的研究

目的:构建草原兔尾鼠卵透明带3 DNA疫苗pVAX1-sig-LTB-lZP3-C3d3进行小鼠的黏膜免疫,增强该疫苗的免疫不育效果。方法:将两种佐剂分子大肠杆菌不耐热肠毒素B亚基和C3d基因通过酶切鉴定,构建重组质粒pVAX1-sig-LTB-lZP3-C3d3,采用RT-PCR和W estern b lot技术,检测其在mRNA和蛋白水平的表达,并通过肌肉注射、滴鼻和灌服3种途径免疫雌性C57BL/6小鼠,通过ELISA检测抗体水平及分型。结果:酶切鉴定,RT-PCR和W estern b lot结果表明重组质粒构建正确并可在mRNA和蛋白水平的表达,ELISA结果表明以重组质粒pVAX1-sig-LTB-lZP3-C3d3免疫诱导的特异性IgG、IgA的水平明显高于对照组(P<0.01)。抗生育实验表明该疫苗免疫的小鼠平均生仔数与对照组相比差异极显著(P<0.01)。结论:构建的草原兔尾鼠卵透明带3DNA疫苗重组质粒pVAX1-sig-LTB-lZP3-C3d3可高效地激发小鼠特异性免疫应答,提高抗生育的效果。     

优化密码子提高草原兔尾鼠ZP3融合蛋白的原核表达

目的:探讨提高草原兔尾鼠(Lagurus lagurus)卵透明带3(LZP3)基因在原核细胞中表达的水平。方法:利用重叠PCR技术,定点突变LZP3基因上3个稀有的密码子簇,将LZP3基因中7个稀有的密码子更换成大肠杆菌(E.coli)最常用的相应密码子。将获得的LZP3突变基因(LZP3m)插入pGEX4T-1中,构建重组表达载体。以重组体转化E.coliBL21(DE3)菌株进行表达。结果:LZP3m基因的表达量比野生型LZP3基因明显提高。结论:通过密码子优化,能显著提高LZP3基因在原核细胞中的表达水平。     

HSP70C融合草原兔尾鼠卵透明带3对小鼠抗生育的研究

目的:探讨分枝杆菌热休克蛋白(HSP70)对草原兔尾鼠卵透明带3(LZP3)免疫不育效果的增强作用.方法:将HSP70全长和C端分别与LZP3融合构建pcD-L-HSP70和pcD-L-HSP70C重组质粒,同时与具有免疫不育功能的pcD-L和pcD-ACLC一起分别免疫NIH小鼠,分别检测重组质粒在机体真核细胞中的表达情况、T细胞增殖及体液免疫水平、抗生育效果、卵巢的病理变化和免疫荧光定位.结果:LZP3构建的重组质粒均能在小鼠肝脏中表达;免疫后能刺激机体T细胞增殖,尤其是pcD-ACLC和 pcD-L-HSP70C免疫组(P<0.01);同时激发机体产生特异性抗体(P<0.05);除pcD-L-HSP70免疫组外其他3种重组质粒均具有抗生育效果(P<0.05),且 pcD-ACLC和pcD-L-HSP70C极明显地降低了小鼠平均窝仔数(P<0.01)且未引发机体产生卵巢炎;直接免疫荧光结果表明在绿色激发光下小鼠卵母细胞的卵透明带均能发出绿色荧光.结论:与pcD-L-HSP70免疫相比,pcD-L-HSP70C能明显地降低小鼠平均窝仔数,这表明分枝杆菌热休克蛋白HSP70C端对增强LZP3免疫不育效果具有明显的基因佐剂功效.     

草原兔尾鼠GST-LZP3融合蛋白的原核表达及其抗体制备

目的 :在原核中表达并纯化草原兔尾鼠卵透明带 3 (zonapellu cida3 ,ZP3 ) ,并以其作为抗原 ,制备抗ZP3的抗血清。方法 :将LZP3基因的核心片段克隆到原核表达载体pGEX 4T 1中。经酶切和序列分析后 ,用重组质粒转化大肠杆菌BL2 1(DE3 ) ,并经丙基β D 硫代半乳糖苷 (IPTG)诱导产生GST LZP3融合蛋白。以纯化的融合蛋白免疫新西兰兔制备抗血清。抗血清的效价及特异性采用ELISA和Westernblot检测。结果 :成功地构建GST LZP3融合蛋白表达载体 ,并在大肠杆菌中高效表达特异性的GST LZP3融合蛋白。以该融合蛋白免疫兔子获得抗GST LZP3融合蛋白的高效价抗血清。结论 :获得原核表达的GST LZP3融合蛋白并制备了兔抗LZP3的抗血清 ,为采用免疫不育技术进行草原兔尾鼠生育控制的研究提供了重要的制剂     

分子佐剂FAI3通过滴鼻途径增强草原兔尾鼠ZP3DNA疫苗抗生育效果

目的:探讨纤维蛋白原/白蛋白/IgG受体3(FAI3)作为分子佐剂,通过滴鼻途径增强免疫反应以提高草原兔尾鼠ZP3 DNA疫苗的抗生育效果。方法:FAI是来自C群链球菌的一种多配基结合蛋白,它能同时结合纤维蛋白原、白蛋白和免疫球蛋白G,FAI的基因片段-fai3(415~702 bp)具有黏膜佐剂的功能。通过构建pcD-fai3重组质粒,用chitosan包裹质粒DNA不育疫苗pcD-Lzp3和质粒pcD-fai3,形成复合物的chi-(pcD-Lzp3+pcD-fai3),以及单独的chi-pcD-Lzp3和chi-pcD-fai3,三种chitosan包裹质粒,分别用这些质粒作为DNA疫苗,分别于第0、14、28、42天,通过滴鼻途径免疫小鼠。间接ELISA检测免疫小鼠血清中抗LZP3特异性IgG,IgG1和IgG2a,同时检测阴道洗液和粪便中的特异性IgA。结果:滴鼻共免疫chi-(pcD-Lzp3+pcD-fai3)诱导产生了高水平的血清IgG和黏膜sIgA,生育率和平均窝仔数显著降低。结论:fai3作为分子佐剂与DNA不育疫苗pcD-Lzp3滴鼻共免疫小鼠,能诱导免疫小鼠产生较强的体液免疫反应和黏膜免疫反应,增强DNA不育疫苗的抗生育效果。     

重组人卵透明带蛋白及其多克隆抗体对精子-透明带结合的影响

为了探讨毕赤酵母表达的重组人卵透明带ZP3蛋白(recombinant human zona pellucida-3 protein,rhZP3)及其多克隆抗体对小鼠和人精卵结合的影响,采用不同浓度的rhZP3以及空白培养液分别处理小鼠精子,然后再与小鼠卵子进行结合实验,观察经过不同处理的精子对透明带黏附及体外受精率的影响;用rhZP3以及空白培养液分别处理人精子,然后再与人卵子进行结合实验,观察经过不同处理的精子对透明带黏附的影响;用抗rhZP3抗体与阴性血清分别处理小鼠和人卵子,再与精子进行结合实验,观察多克隆抗体对精子粘附以及小鼠体外受精率的影响。实验结果表明,rhZP3和抗rhZP3多克隆抗体既能抑制人的体外精卵结合,也能抑制小鼠体外精卵结合,提示rhZP3具有天然透明带的特性,有发展成避孕疫苗和作为检测透明带抗体检测试剂的可能。     

3-羟酰基辅酶A脱氢酶在大肠杆菌中的表达及抗血清的制备

目的：克隆并在大肠杆菌中表达3-羟酰基辅酶A脱氢酶（3-hydroxyacyl-CoA dehydrogenase，HCDH）并制备其抗血清。方法：通过PCR方法扩增HCDH基因片段，经DNA测序证实后克隆到原核表达载体pMAL-P2X，重组质粒转化大肠杆菌DE3，IPTG诱导表达，SDS-PAGE分析表达结果，经麦芽糖树脂亲和纯化的蛋白皮下注射免疫獭兔，琼扩和ELISA检测抗血清的效价，Western blot检测其特异性。结果：PCR扩增得到786bp的目的片段，序列测定证实与GeneBank上登录的序列一致；成功构建了HCDH基因片段的原核表达载体；在详细探索表达条件的基础上，成功诱导HCDH的表达，并制备了相应的抗血清。结论：检测HCDH蛋白在表达水平上的变化及为HCDH基因表达调控的进一步研究奠定了基础。     

Immunoreactivity and in-vitro effect on human sperm-egg binding of antibodies against peptides corresponding to bonnet monkey zona pellucida-3 glycoprotein

The following synthetic peptides were made as immunogens for development of a zona-based contraceptive vaccine: P1, KQPFWLLQGGASRAETSVQPVLVE [amino acids (aa) 23-45 with an additional K at the N-terminus]; P2, FSEEKLVFSLRLMEENC (aa 164-179 with an additional C at the C-terminus and T170 replaced by V); and P3, CSFSKSSNSWFPVEGPADICQCC (aa 300-322). The aa are numbered on the basis of bonnet monkey ZP3 precursor protein. Antibodies against an additional peptide P4, KGDCGTPSHSRRQPHVVSQWSRSA (aa 324-347), significantly inhibits human sperm-oocyte binding. In addition, antibodies against cocktail of peptide-diphtheria toxoid conjugates also significantly inhibit the binding of spermatozoa to zona pellucida in a hemizona assay. These results will further help in the design of an immunocontraceptive vaccine based upon synthetic peptides corresponding to ZP3.      

Reactivity of a recombinant rubella E1 antigen expressed inE. coli

Summary The E1 nucleic acid sequence of rubella virus strain Judith (RJ) has been cloned into anE. coli expression vector LB03. The reactivity of the expressed unglycosylated antigen (E1J) was compared with its glycosylated counterpart in native virus (RJ) usind rabbit and human sera. Rabbit antisera raised against RJ and E1J reacted differently with wild type, RJ (laboratory strain) and RA27/3 (vaccine virus) strains in a kinetic neutralisation test. Reciprocally, human post RA27/3 vaccination sera were also found to differ from post infection or post re-infection sera in their reactivity with RJ and E1J antigens. Our observations suggest that E1, in the conformation adopted in the RA27/3 virion may have unique antigenic properties.     

猪卵透明带—4蛋白在大肠杆菌中高表达的研究

目的：在大肠杆菌中高水平地直接表达目的蛋白,方法：用PCR方法从猪卵透明带－1(pZP1)cDNA中扩增pZP4目的基因片段,通过在其5′端和3′端引入的双酶切位点将该完整的目的cDNA定向插入pBV221表达质粒PRPL启动因子下游的多克隆区。结果：此重组表达质粒转化宿主工程菌后经热诱导培养,可在SDS－PAGE凝胶上得到特异性的高表达蛋白条带,而且它在Western blot鉴定实验中能被鼠抗pZP4的单克隆抗体17D3和兔抗pZP IgGs识别。结论：成功地在大肠杆菌高效表达了可被17D3单克隆抗体及ZP多克隆体识别的ZP4。     

重组人Flt3配体在大肠杆菌中的高效表达和生物学活性的鉴定

根据天然的人Flt3配体(Flt3ligand,FL)基因和遵循适合大肠杆菌BL21(DE3)表达体系表达条件及不改变FL天然蛋白氨基酸序列的原则,对其进行改造,所获得的FL功能片段(FL134)克隆入PET30a表达载体,在C端融合了6个His,继而在大肠杆菌中诱导表达。结果表明重组蛋白rhFL134以包涵体形式在大肠杆菌(BL21)中可得到较高水平的表达。通过蛋白的变性、复性和HiTrapTM亲和层析获得了纯度达92%以上的rhFL134重组蛋白。体外的活性实验显示,重组蛋白rhFL134具有显著的促小鼠骨髓细胞的集落形成和扩增人脐带血中的CD34+细胞的生物学活性。     

A two step purification of recombinant human interleukin-1 beta expressed in E. coli

Recombinant human interleukin-1 beta has been expressed in high yield using E. coli with a cDNA clone obtained from SKhep1RNA. The rIL-1 beta is purified to apparent homogeneity using freeze-thaw extractions followed by hydrophobic interaction chromatography over phenyl Sepharose. The procedure can provide pure rIL-1 beta (up to 15 mg per liter of E. coli culture) without the use of denaturants and if desired, in the absence of column chromatographic steps. Purity is defined by the presence of a single band on 1-D polyacrylamide gels and a single spot on 2-D polyacrylamide gels. The purified protein exhibits a biological activity of 1 x 10(7) units/mg in a fibroblast proliferation assay and is shown to cross-react with rabbit anti-human IL-1 beta sera.     

人LC3蛋白在大肠杆菌中的表达纯化及其抗体的制备与鉴定

目的:制备兔抗人LC3的多克隆抗体,为细胞自噬的研究提供有用的检测工具。方法:构建pET32a(+)LC3的原核细胞表达载体并进行序列鉴定,大肠杆菌BL21(DE3)中诱导表达LC3蛋白,SP-Sepharose XL柱纯化;纯化的人LC3蛋白免疫兔制备抗血清,通过LC3蛋白偶联的Sepharose 4B(LC3-Sepharose 4B)亲和层析纯化兔抗人LC3的多克隆抗体,以间接ELISA和Western blot鉴定其特异性。结果:成功实现了LC3蛋白在大肠杆菌中的高效表达和纯化,并用质谱分析证明表达蛋白相对分子质量(Mr)为15 500。制备的兔抗人LC3多克隆抗体具有很高的特异性,与大肠杆菌成分无交叉反应。Western blot能够特异检测自噬过程中LC3Ⅰ型和Ⅱ型蛋白,观察到自噬发生时LC3Ⅰ向LC3Ⅱ的转化。结论:获得了重组人LC3蛋白,成功地制备了该分子的特异性多克隆抗体,为细胞自噬的检测提供了有用的探针。     

Expression, purification and identification of recombinant mouse interleukin 21 protein in E. coli

Interleukin 21 （IL-21） is a novel type I cytokine that is significantly homologous to IL-2, IL-4 and IL-15. Its receptor complex contains γc chain which is also a component of receptors for IL-2, IL-4, IL-7, IL-9 and IL-15, so there may be overlapping or relevancies in their biological functions. IL-21 is capable of co-stimulating mature T cells, B cells, NK cells, and of stimulating CD16 expression on the surface of NK cells to induce ADCC in innate immune response. It can also strengthen the anti-tumor effect of the cellular immunity, especially v/a enhancing the activities of NK and antigen specific CTL cells. Thus, IL-21 is a potential useful therapeutic molecule for immunotherapy of malignancies, by eliciting innate and adaptive anti-tumor immune responses in tumor-bearing hosts. In order to study the biological functions of IL-21, we constructed a mIL-21 prokaryotic expression plasmid and expressed the recombinant mIL-21 protein in E. coli in present study. The recombinant plasmid pET28a/mIL-21 with a carboxyl terminal His-tag was subcloned from the pcDNA3.1/mIL-21 and expressed in E. coli. The induced protein was detected by SDS-PAGE, and identified by Western-blot assay with anti-mIL-21 antibody. The recombinant protein was purified v/a Ni^＋ affinity chromatography, and renatured with GSH/GSSG system. Our mouse T cell proliferation experiment showed that the recombinant mIL-21 protein could enhance the mouse T cell proliferation either by itself alone or in the presence of Con A.     

Purification of the Sendai virus nonstructural C protein expressed in E. coli,and preparation of antiserum against C protein

Summary An expression plasmid, ptac-C, was constructed by inserting the cDNA of the coding region of the Sendai virus nonstructural C protein downstream of the tac promoter ofE. coli expression plasmid ptac12-Bam. A new protein produced inE. coli after induction was purified to near homogeneity. The purified protein was found to be identical with the C protein predicted from the C gene cDNA in molecular weight, isoelectric point, amino acid composition, and the amino acid sequence at the N-terminal of the protein as well as those of several fragments obtained on V8 protease digestion. Antiserum raised against the purified protein specifically reacted with the C protein in infected cells. Using this antiserum, the localization of the C protein in infected cells was examined by immunofluorescence, which revealed that it appeared in the cytoplasm but not in nuclei.