AAV9衣壳蛋白VP的原核表达、纯化及多克隆抗体制备

目的:表达腺相关病毒(AAV)9型衣壳蛋白VP,制备抗VP蛋白的多克隆抗体。方法:使用PCR技术从AAV9病毒包装质粒中扩增AAV9衣壳蛋白VP,同源重组法构建衣壳蛋白表达质粒pET30a-AAV9-VP。质粒转化表达菌E.coli BL21(DE3)后,IPTG诱导目的蛋白VP表达,亲和层析法纯化VP蛋白,将纯化后的蛋白免疫日本大耳白兔,3次免疫后获得针对VP蛋白的兔多克隆抗体。以Western blot和细胞免疫荧光法检测抗体的应用,ELISA检测所得抗体的效价。结果:成功构建pET30a-AAV9-VP表达质粒,在大肠杆菌BL21中,IPTG可诱导VP蛋白表达。亲和层析法纯化获得高纯度的VP蛋白。该蛋白在日本大耳兔体内能够诱导产生多克隆抗体,ELISA检测抗血清效价达到1∶512 000。结论:成功原核表达和纯化AAV9衣壳蛋白VP并制备了兔多克隆抗体。制备的抗AAV9 VP抗体能够特异性识别和结合AAV衣壳蛋白,并可有效用于Western blot和细胞免疫荧光分析,为后续深入研究AAV9在基因治疗中的作用及AAV9载体开发提供了研究基础。

水稻锌指蛋白OsRZ3基因克隆及融合蛋白的原核表达、纯化

根据NCBI登录的水稻锌指蛋白基因(NO.AK062094,OsRZ3 gene)设计特异引物,通过RT-PCR克隆该基因cDNA全长序列,核苷酸测序并比对氨基酸同源性表明具有C2HC-锌指结构域、分析预测蛋白质分子量为34kD和等电点为9.05;拟南芥原生质体亚细胞定位检测表明其在细胞核。构建pGEX6P-3::OsRZ3融合载体,电转化的大肠杆菌BL21(DE3)菌株经1 mmol·L-1IPTG小量诱导表达,SDS-pAGEX蛋白电泳表明60 kD融合蛋白4 h最大;在LB液体培养中0.5 mmol·L-1IPTG过夜大量诱导表达,通过GST吸附柱层析获得大量包涵体纯化蛋白,1L菌液可诱导纯化到浓度1.58 mg·mL-1包涵体融合蛋白。所构建的融合蛋白原核表达系统能有效表达pGEX6P-3::OsRZ3融合蛋白,0.5 mmol·L-1IPTG大量诱导,通过GST柱吸附获得包涵体纯化蛋白,为作进一步的抗体制备和染色质免疫共沉淀等DNA结合的特性研究准备了基础。

Smarcad1重组蛋白表达及多克隆抗体验证

目的:建立稳定的实验室多克隆抗体制备流程,选择Smarcad1作为目的基因进行重组蛋白表达纯化与多克隆抗体制备,并对其进行特异性验证。方法:以本实验室的pcDNA3.1-Flag-Smarcad1质粒作为模板,构建pET-16b-Smarcad1-F1原核表达质粒。表达Smarcad1-F1蛋白,并纯化。利用蛋白质免疫印迹,蛋白质免疫共沉淀和免疫荧光实验检测抗体特异性。结果:PCR鉴定结果显示,成功构建pET-16b-Smarcad1-F1表达质粒。蛋白表达纯化实验中,考马斯亮蓝染色检测,相较于诱导前,加入IPTG后Smarcad1蛋白成功诱导表达,且纯化出分子量为27 kD的目的蛋白。Western印迹证明,制备的抗体能够特异性识别内源性Smarcad1蛋白。蛋白质免疫共沉淀实验显示,相较于对照组,制备的抗体能够与Smarcad1蛋白结合。免疫荧光实验检测所制备抗体的特异性,结果显示制备的抗体可以特异性识别内源性Smarcad1蛋白。结论:成功构建了Smarcad1原核表达质粒,利用原核蛋白表达纯化出Smarcad1蛋白,利用该蛋白制备的抗体可以特异性识别内源性Smarcad1蛋白,为后续原核表达蛋白及多克隆抗体制备提供思路。

Establishment of an Indirect ELISA with the Major Epitope Domain of ApxⅡ of Actinobacillus pleuropneumoniae Expressed in Prokaryotic Cells

[Objective] This study aimed to develop an indirect ELISA to detect the antibodies against Actinobacil us pleuropneumoniae （APP） using the recombinant ApxⅡA1 protein expressed in prokaryotic cells as the antigen. [Method] The major epi-tope domain of ApxⅡ was cloned into the prokaryotic expression vector pET-28a （＋） to obtain the recombinant plasmid pET-ApxⅡA1, which was then transformed into E. coli BL21 （DE3） for expression. The immunogenicity of the recombinant pro-tein was analyzed by western-blotting. After that, the purified recombinant protein was used as the coating antigen in the indirect ELISA for detecting the antibodies against APP. Final y, the concentration of coated antigen and the dilution of serum were optimized. [Result] Proved by enzyme digestion and sequencing, the recombi-nant plasmid pET-ApxⅡA1 was constructed successful y. The recombinant protein was highly expressed in prokaryotic cells, and Western-blotting analysis showed that it was recognized specifical y by positive serum of APP. The indirect ELISA could detect the antibody against APP with the purified recombinant protein as the coating antigen. The optimal concentration of coated antigen was 1.23 μg/ml and the opti-mal dilution of serum was 1：100. Compared with a commercial ELISA kit detecting antibody against ApxⅣ, the coincidence rate of the indirect ELISA was 90.4%. [Conclusion] Our results indicated that the indirect ELISA is sensitive and specific, and suitable for evaluating the effect of APP vaccine and epidemiological surveys.

Gene cloning and prokaryotic expression of recombinant flagellin A from Vibrio parahaemolyticus

The Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals. Bacteria flagellins play an important role during infection and induction of the host immune response. Thus, flagellin proteins are an ideal target for vaccines. We amplified the complete flagellin subunit gene （tTaA） from V. parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 （DE3） cells. The gene coded for a protein that was 62.78 kDa. We purified and characterized the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting, respectively. Our results provide a basis for further studies into the utility of the FlaA protein as a vaccine candidate against infection by Vibrio parahaemolyticus. In addition, the purified FlaA protein can he used for further functional and structural studies.

Expression and Purification of the Major Outer Membrane Protein of Chlamydia Trachomatis in Prokaryotic Cell

To clone and construct the recombinant plasmid containing the major outer membrane protein (MOMP) gene of Chlamydia trachomatis (C.trachomatis) and to express the fusion protein in E.coli BL21, the MOMP gene was amplified by polymerase chain reaction (PCR) from genome of C.trachomatis serovar D. The fragment was cloned into the prokaryotic expression vector pET-22b(+) after digestion with BamHⅠ and NotⅠ and transformed into E.coli XL1-Blue. Recombinants were selected by enzyme digestion and sequencing and the recombinant plasmid with MOMP gene was then transformed into E.coli BL21 with IPTG to express the target gene. The expression recombinant proteins were purified by Ni-NTA affinity chromatography, and identified by SDS-PAGE and Western blot. It was found that a 1.2?kb MOMP gene was isolated. The DNA sequence of MOMP was found to be just the same as the sequence published by GenBank. A recombinant plasmid containing MOMP gene was constructed to express the fusion proteins in E.coli. SDS-PAGE analysis showed that the relative molecular weight of the recombinant protein was about 47?kDa that was consistent with the theoretical predicted value, and the specificity of the expressed protein was conformed by Western blot. It concluded that the MOMP gene could be expressed in the prokaryotic system, by which it provided the foundation for the future studies on the biological activities of C.trachomatis and for the development of vaccine against this pathogen.