Guang-Li Wang, Wei Liang, Ling-Yun Li, Song-Quan Wu
Medical College of Lishui University, Zhejiang Province, China.
The People's Hospital of Lishui City, Lishui City, Zhejiang Province, China.
Ann Clin Lab Sci. 2018 May;48(3):323-327.
To clone, express, purify the Per a 4 gene encoding an allergen of and prepare monoclonal antibodies against the recombinant allergen.
The total RNA was extracted from , and the target gene was amplified by RT-PCR and cloned into pMD18-T vector. After being confirmed by nucleotide sequencing, the gene was then inserted into pGEX-3X to construct the express vector pGEX-3X-Per a 4. Further, the pGEX-3X-Per a 4 was transformed into BL21 (DE3), and induced for expression by IPTG. By affinity chromatography, the recombinant allergen was purified and identified by SDS-PAGE and Western blotting. The BALB/c mouse was immunized with the recombinant allergen to prepare the specific monoclonal antibodies, which was then identified by co-immunoprecipitatin and western blotting.
The full-length cDNA encoding Per a 4 of was obtained with 552 bp in length, which had 99.4% similarity with the reference sequence (GenBank AY792948). The constructed vector pGEX-3X-Per a 4 was transformed in BL21 (DE3), expressed with the induction of IPTG. By SDS-PAGE, a band of about 49 KD was present. Further, the western-blotting showed that the prepared monoclonal antibodies can bind the serum antibodies in patients allergic to .
Both the recombinant allergen Per a 4 and its monoclonal antibodies were obtained.
克隆、表达、纯化编码[某种过敏原名称]的Per a 4基因，并制备针对该重组过敏原的单克隆抗体。
从[某种生物名称]中提取总RNA，通过RT-PCR扩增目标基因并克隆至pMD18-T载体。经核苷酸测序确认后，将该基因插入pGEX-3X构建表达载体pGEX-3X-Per a 4。进一步将pGEX-3X-Per a 4转化至大肠杆菌BL21(DE3)，用IPTG诱导表达。通过亲和层析纯化重组过敏原，经SDS-PAGE和Western印迹鉴定。用重组过敏原免疫BALB/c小鼠制备特异性单克隆抗体，再经共免疫沉淀和Western印迹鉴定。
获得了编码[某种生物名称]Per a 4的全长cDNA，长度为552 bp，与参考序列（GenBank AY792948）相似度为99.4%。构建的载体pGEX-3X-Per a 4转化至大肠杆菌BL21(DE3)，经IPTG诱导表达。SDS-PAGE显示有一条约49 KD的条带。此外，Western印迹表明制备的单克隆抗体能与对[某种生物名称]过敏患者的血清抗体结合。
获得了重组过敏原Per a 4及其单克隆抗体。
