• <mark id="l0x4h"></mark>

      <u id="l0x4h"></u>
        <ins id="l0x4h"></ins>

      1. <bdo id="l0x4h"></bdo>
      2. 抗C反應蛋白單域抗體噬菌體庫的構建與初步鑒定
        DOI:
        作者:
        作者單位:

        作者簡介:

        通訊作者:

        中圖分類號:

        基金項目:

        國家自然科學基金面上項目(31873006; 31572556); 烏魯木齊市科技計劃項目(P16130001); 省部共建綿羊遺傳改良與健康養殖國家重點實驗室優秀中青年人才培養引導計劃專項(SKLSGIHP2017A03)


        Construction and identification of phage library of single domain antibody against C-reactive protein
        Author:
        Affiliation:

        Fund Project:

        • 摘要
        • |
        • 圖/表
        • |
        • 訪問統計
        • |
        • 參考文獻
        • |
        • 相似文獻
        • |
        • 引證文獻
        • |
        • 資源附件
        • |
        • 文章評論
          摘要:

          以CRP為免疫原免疫雙峰駝后分離外周血淋巴細胞,提取總RNA后經逆轉錄制備cDNA,通過巢式PCR實驗獲得目的基因片段并克隆至噬菌體T7載體臂上,構建原始噬菌體庫。隨后進行4輪生物淘選,通過Phage-ELISA鑒定淘選后的噬菌體庫,BamHⅠ、HindⅢ 雙酶切構建了pET-28a與陽性克隆的重組質粒,轉化入BL21菌株中進行低溫誘導表達。經過His-鎳離子親和層析柱純化獲得單域抗體,然后再用間接ELISA鑒定單域抗體與CRP的反應性。研究結果顯示,試驗成功構建了庫容為1.08×108 cfu的抗CRP單域抗體噬菌體庫,基因插入率為96.43%(27/28)。生物淘選獲得了4株與CRP特異性結合的陽性克隆,4株陽性克隆表達載體構建成功,并在BL21細胞中成功表達單域抗體,分子質量約為22 ku。經ELISA鑒定,4個抗體均顯示出高親和力與特異性,其中V2與V3的抗體效價高達1∶3 200,證明該抗體能夠適用于實際檢測方法開發。

          Abstract:

          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.

          參考文獻
          相似文獻
          引證文獻
        引用本文

        張齊,張星星,鐘發鋼,王金泉,張小鶯.抗C反應蛋白單域抗體噬菌體庫的構建與初步鑒定[J].華中農業大學學報,2020,39(4):93-98

        復制
        分享
        文章指標
        • 點擊次數:
        • 下載次數:
        • HTML閱讀次數:
        • 引用次數:
        歷史
        • 收稿日期:2019-12-04
        • 最后修改日期:
        • 錄用日期:
        • 在線發布日期: 2020-07-30
        • 出版日期:
        牌九游戏