国家自然科学基金面上项目（31873006; 31572556）； 乌鲁木齐市科技计划项目（P16130001）； 省部共建绵羊遗传改良与健康养殖国家重点实验室优秀中青年人才培养引导计划专项（SKLSGIHP2017A03）
以CRP为免疫原免疫双峰驼后分离外周血淋巴细胞，提取总RNA后经逆转录制备cDNA，通过巢式PCR实验获得目的基因片段并克隆至噬菌体T7载体臂上，构建原始噬菌体库。随后进行4轮生物淘选，通过Phage-ELISA鉴定淘选后的噬菌体库，BamHⅠ、HindⅢ 双酶切构建了pET-28a与阳性克隆的重组质粒，转化入BL21菌株中进行低温诱导表达。经过His-镍离子亲和层析柱纯化获得单域抗体，然后再用间接ELISA鉴定单域抗体与CRP的反应性。研究结果显示，试验成功构建了库容为1.08×108 cfu的抗CRP单域抗体噬菌体库，基因插入率为96.43%（27/28）。生物淘选获得了4株与CRP特异性结合的阳性克隆，4株阳性克隆表达载体构建成功，并在BL21细胞中成功表达单域抗体，分子质量约为22 ku。经ELISA鉴定，4个抗体均显示出高亲和力与特异性，其中V2与V3的抗体效价高达1∶3 200，证明该抗体能够适用于实际检测方法开发。
After immunizing Bactrian camel with CRP as immunogen,the peripheral blood lymphocytes were isolated,the total RNA was extracted,and then the cDNA was obtained by reverse transcription. The target gene fragment was obtained by nested PCR and cloned on the arm of T7 vector of phage to construct the original phage library. After four rounds of biological panning,the phage library after panning was identified by Phage-ELISA. The recombinant plasmids of pET-28a and positive clones were constructed by BamH Ⅰ and HindⅢ double enzyme digestion and transformed into BL21 strain for low-temperature induction expression. The single domain antibody was purified by His-Ni affinity chromatography,and then the reactivity between the single domain antibody and CRP was identified by indirect ELISA. The results showed that the phage library with a capacity of 1.08×108 cfu was successfully constructed,and the gene insertion rate was 96.43% (27/28). Four positive clones were successfully constructed and expressed in BL21 cells. The molecular weight was about 22 ku. By ELISA,the four antibodies showed high affinity and specificity. The titer of V2 and V3 was 1∶3 200,which proved that the antibody could be applied to the development of practical detection methods.