Preparation of Polyclonal Antibody Against c-Myc Protein in Sus scrofa
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摘要:
目的 克隆猪c-Myc基因CDS序列并构建其原核表达载体，同时纯化c-Myc蛋白并以此为抗原制备多克隆抗体，为进一步研究猪圆环病毒2型（Porcine circovirus type 2，PCV2）Rep蛋白与宿主c-Myc蛋白之间的交互作用奠定基础。 方法 根据猪c-Myc全基因序列（GenBank No. NM_001005154）设计引物，以反转录PCR方法扩增c-Myc基因CDS序列，经Nde I/Xho I双酶切后定向克隆至原核表达载体pET-30a（+）；转化至Arctic-ExpressTM大肠杆菌后诱导表达；表达产物经变性、复性和His-Band Ni+层析柱亲和纯化后免疫新西兰白兔。抗原亲和层析法纯化抗体后，用间接ELISA法测定其效价，用Western blot检测其特异性。 结果 经检测，克隆产生的猪c-Myc基因CDS序列长度为1 359 bp，c-Myc-His重组蛋白主要以包涵体形式出现，分子质量约为63 kDa，由此蛋白制备的多抗效价可以达到1∶1 093 500，并能特异性识别猪c-Myc蛋白和重组蛋白c-Myc-His。 结论 本研究成功制备了猪c-Myc蛋白多克隆抗体，为研究猪c-Myc蛋白功能和其与PCV2 Rep蛋白之间的相互作用奠定了良好基础。 Abstract:Objective To facilitate studies on the functions of the porcine circovirus type 2 Rep protein and its interaction with the host c-Myc protein, a polyclonal antibody against the virus was prepared. Method According to the complete sequence of porcine c-Myc (GenBank No. NM_001005154), primers were designed to amplify the CDS sequence of the gene by reverse transcription PCR. The CDS was double-digested by Nde I/Xho I enzymes to construct the prokaryotic expression vector pET-30a(+) and transformed into bacterium Arctic-ExpressTM (Escherichia coli) for induction and expression. The obtained products were denatured, re-natured, and affinity purified by His-band Ni+ chromatography as the antigen to prepare polyclonal antibody by immunizing New Zealand white rabbits. The resulting antibody was purified by the antigen affinity purification chromatography, and the titer and specificity determined by indirect ELISA and western blot. Result The cloned CDS-sequence of porcine c-Myc was 1359 bp. The recombinant protein of c-Myc-His was mainly in the form of inclusion bodies with a molecular weight of approximately 63 kDa. The titer of the polyclonal antibody prepared from the protein was greater than 1∶1093500. It could specifically recognize the target proteins including recombinant c-Myc-His and porcine c-Myc proteins. Conclusion The target polyclonal antibody against the porcine c-Myc protein was successfully obtained for further studies on the functions of the protein as well as its interactions with the PCV2 Rep protein. -
图 2 猪c-Myc 蛋白的亲/疏水性分析
Figure 2. Hydrophilicity/hydrophobicity of porcine c-Myc protein
图 3 重组表达质粒pET-c-Myc的双酶切鉴定
M：DNA Marker；1：pET-c-Myc质粒对照；2：pET-c-Myc质粒双酶切。
Figure 3. Identification of recombinant expression plasmid pET-c-Myc by double digestion
M: DNA marker; 1: control of pET-c-Myc plasmid; 2: double-digested pET-c-Myc plasmid.
图 4 重组蛋白c-Myc-His表达的SDS-PAGE分析
M：蛋白质分子质量标准；1：经pET-30a(+)诱导（空载）的产物；2：未经IPTG诱导的pET-c-Myc产物；3：经IPTG诱导的pET-c-Myc产物；4：IPTG诱导的pET-c-Myc上清液；5：IPTG诱导的pET-c-Myc沉淀。
Figure 4. Expression and solubility of c-Myc-His recombinant protein determined by SDS-PAGE
M: Protein molecular weight marker; 1: product from pET-30a(+) (empty load) induced by IPTG; 2: product from uninduced pET-c-Myc; 3: product from pET-c-Myc induced by IPTG; 4: supernatant from pET-c-Myc induced by IPTG; 5: precipitate from pET-c-Myc induced by IPTG.
图 5 重组蛋白c-Myc-His纯化的SDS-PAGE分析
M：蛋白质分子质量标准；1：经 IPTG诱导的菌体总蛋白；2：Ni-IDA Washing-Buffer 冲洗后的流出液；3~4：Ni-IDA Elution-Buffer洗脱后的流出液。
Figure 5. SDS-PAGE analysis on purified c-Myc-His recombinant protein
M: Protein molecular weight marker; 1: total protein of bacteria induced by IPTG; 2: effluents after washing by Ni-IDA washing-buffer; 3—4: effluents after washing by Ni-IDA elution-buffer.
图 6 猪c-Myc蛋白多克隆抗体特异性的Western blot分析
M：蛋白标准；1：重组蛋白His-Hsp40；2：重组蛋白c-Myc-His。
Figure 6. Specificity of polyclonal antibody against porcine c-Myc protein determined by western blot
M: Protein marker; 1: recombinant protein His-Hsp40; 2: recombinant protein c-Myc-His.
图 7 猪c-Myc蛋白多克隆抗体与猪c-Myc蛋白的间接免疫荧光反应(200×)
A. pcDNA3.1(＋)转染的PAM细胞；B. pcDNA-c-Myc转染的PAM细胞。
Figure 7. Indirect immunofluorescence assay on polyclonal antibody and porcine c-Myc protein (200×)
A. pcDNA3.1(+) transfected PAM cells; B: pcDNA3.1-c-Myc transfected PAM cells.
表 1 间接ELISA法测定猪c-Myc蛋白多克隆抗体的效价
Table 1. Indirect ELISA determination on polyclonal antibody titer against porcine c-Myc protein
抗体稀释度
Antibody dilutionOD450 nm 1∶500 3.708 1∶1500 3.407 1∶4500 3.399 1∶13500 3.368 1∶40500 3.231 1∶121500 2.629 1∶364500 1.267 1∶1093500 0.589 1∶3280500 0.328 1∶9841500 0.243 1∶29524500 0.202 空白 Mock 0.190 滴度 Titer ＞1093500 
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