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Establishment of primary chicken embryo myoblast cell culture, antigenic epitopes prediction and production of anti activin receptor type IIB polyclonal antibody in chicken
Animal Biotechnology ( IF 3.7 ) Pub Date : 2021-01-24 , DOI: 10.1080/10495398.2020.1870483
Pothanaboyina Guru Vishnu 1 , T K Bhattacharya 2 , Devara Divya 3 , Athe Rajendra Prasad 4 , N Govardhan Sagar 4
Affiliation  

Abstract

The detection of activin receptor typeIIB (ACTRIIB) protein, a prominent negative muscle growth regulator has paramount value in augmenting growth traits through molecular breeding schemes in chicken. The study was formulated to establish primary chicken embryo myoblast culture (CEM) using 9th and 18th day chick embryos and to develop antibodies for immunodetection of ACTRIIB protein. The physicochemical and structural attributes of the ACTRIIB sequence were evaluated to identify substantial antigenic regions. The ACTRIIB sequence was transfected into CEM and expressed protein was injected subcutaneously into rats to produce hyperimmune serum. The average propensity of protein sequence for beta turns, surface accessibility, chain flexibility, antigenicity, hydrophilicity and linear epitopes was 0.978, 1.000, 0.991, 1.038, 1.258 and 0.512, respectively. The 9th day CEM exhibited confluency (80–90%) earlier than the 18th day. The expression of myogenic regulatory factors in 9th day myoblasts was higher than the 18th day by 7.28, 5.16, 6.28 and 6.93 folds for MYF5, MRF4, MYOG and MYOD, respectively. The ACTRIIB mRNA was downregulated by 2.54 folds on the 9th day compared to the 18th day myoblasts and protein varied significantly between 9th and 18th day myoblasts. The CEM culture can be harnessed unequivocally to investigate molecular mechanisms underlying muscle growth besides raising antibodies.



中文翻译:

原代鸡胚成肌细胞培养的建立、抗原表位预测及抗激活素受体ⅡB型多克隆抗体的制备

摘要

激活素受体 typeIIB ( ACTRIIB)蛋白的检测是一种显着的负性肌肉生长调节剂,对于通过分子育种计划增强鸡的生长性状具有至关重要的价值。该研究旨在使用第 9 天和第 18 天的鸡胚建立原代鸡胚成肌细胞培养物 (CEM),并开发用于ACTRIIB蛋白免疫检测的抗体。评估了ACTRIIB序列的物理化学和结构属性,以确定实质性抗原区域。益智_序列被转染到 CEM 中,表达的蛋白质被皮下注射到大鼠体内以产生超免疫血清。蛋白质序列对β转角、表面可及性、链柔性、抗原性、亲水性和线性表位的平均倾向分别为0.978、1.000、0.991、1.038、1.258和0.512。第 9 天 CEM 比第 18 天更早出现融合 (80–90%)。对于MYF5、MRF4、MYOGMYOD ,第 9 天成肌细胞中生肌调节因子的表达比第 18 天高 7.28、5.16、6.28 和 6.93 倍。益智_与第 18 天的成肌细胞相比,第 9 天的 mRNA 下调了 2.54 倍,并且第 9 天和第 18 天的成肌细胞之间的蛋白质差异显着。除了提高抗体外,CEM 培养物还可以明确地用于研究肌肉生长的分子机制。

更新日期:2021-01-24
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