Identification of cocoonase and cocoonase like protein using polyclonal antibody of Antheraea mylitta cocoonase

IF 2 4区生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Biotechnology Letters Pub Date : 2023-12-18 DOI:10.1007/s10529-023-03447-y

Aruna Rani, Dev Mani Pandey, Jay Prakash Pandey

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Abstract

Background

Cocoonase is a proteolytic enzyme released by silk moths during pupal adult emergence. Without damaging the silk fibroin, this enzyme dissolves the shell of the tasar cocoon by exclusively targeting the protein sericin. Prior to this study, there was no available antibody against Antheraea mylitta cocoonase to identify or screen out similar variants or cocoonase like protein.

Results

In the present study, naturally secreted A. mylitta cocoonase was purified and used to immunize New Zealand white rabbits. The developed polyclonal antibody of cocoonase was purified and its specific interaction with cocoonase was determined using Indirect ELISA. The confirmation of its specificity and immuno-reactivity was evaluated by western blot using native cocoonase of tasar silkworm A. mylitta. The efficacy and specificity of the polyclonal antibody were further verified and confirmed by western blot which was performed to detect ten different ecotypes of A. mylitta cocoonase.

Conclusion

The developed antibody successfully detected the cocoonase of different ecotypes. Thus, in future this antibody can serve as one of the molecular detection method for cocoonase and cocoonase-like proteins.

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使用多克隆抗体鉴定蚁蚕茧酶和类茧酶蛋白

背景蚕茧酶是一种蛋白水解酶，由丝蛾在蛹成虫蜕皮时释放。在不破坏蚕丝纤维蛋白的情况下，这种酶通过专门针对丝胶蛋白来溶解蚕茧的外壳。在本研究之前，还没有针对 Antheraea mylitta 茧蛋白酶的抗体来鉴定或筛选出类似的变体或类似茧蛋白酶的蛋白质。纯化了所开发的茧酶多克隆抗体，并使用间接酶联免疫吸附法测定了其与茧酶的特异性相互作用。通过使用塔沙蚕 A. mylitta 的原生茧酶进行 Western 印迹，对其特异性和免疫反应性进行了评估。多克隆抗体的有效性和特异性通过检测十种不同生态型的 A. mylitta 蚕茧酶的 Western 印迹得到了进一步验证和确认。因此，该抗体将来可作为茧酶和茧酶样蛋白的分子检测方法之一。

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来源期刊

Biotechnology Letters 工程技术-生物工程与应用微生物

CiteScore

5.90

自引率

3.70%

发文量

108

审稿时长

1.2 months

期刊介绍： Biotechnology Letters is the world’s leading rapid-publication primary journal dedicated to biotechnology as a whole – that is to topics relating to actual or potential applications of biological reactions affected by microbial, plant or animal cells and biocatalysts derived from them. All relevant aspects of molecular biology, genetics and cell biochemistry, of process and reactor design, of pre- and post-treatment steps, and of manufacturing or service operations are therefore included. Contributions from industrial and academic laboratories are equally welcome. We also welcome contributions covering biotechnological aspects of regenerative medicine and biomaterials and also cancer biotechnology. Criteria for the acceptance of papers relate to our aim of publishing useful and informative results that will be of value to other workers in related fields. The emphasis is very much on novelty and immediacy in order to justify rapid publication of authors’ results. It should be noted, however, that we do not normally publish papers (but this is not absolute) that deal with unidentified consortia of microorganisms (e.g. as in activated sludge) as these results may not be easily reproducible in other laboratories. Papers describing the isolation and identification of microorganisms are not regarded as appropriate but such information can be appended as supporting information to a paper. Papers dealing with simple process development are usually considered to lack sufficient novelty or interest to warrant publication.