Preparation and Detection of Polyclonal Antibodies of Key Factors for Muscle Development of Yellow River Carp (Cyprinus carpio haematopterus)

YUN Yinghao; SONG Dongying; HE Zijie; MI Jiali; WANG Luming; NIE Guoxing

doi:10.3969/j.issn.1006-267x.2022.03.052

2022 , Vol. 34 >Issue 3: 1931 - 1941

DOI: https://doi.org/10.3969/j.issn.1006-267x.2022.03.052

RESEARCH PAPER

Preparation and Detection of Polyclonal Antibodies of Key Factors for Muscle Development of Yellow River Carp (Cyprinus carpio haematopterus)

YUN Yinghao ,
SONG Dongying ,
HE Zijie ,
MI Jiali ,
WANG Luming ,
NIE Guoxing

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1. College of Fisheries, Henan Normal University, Xinxiang 453007, China;
2. School of Marine Sciences, Ningbo University, Ningbo 315211, China

Received date: 2021-08-03

Online published: 2022-03-14

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Abstract

Most studies on muscle development of aquaculture animals on transcription level, and few studies on protein level. This limitation restricts in-depth research on muscle development and growth of aquaculture animals. In this study, the white muscle of Yellow River carp (Cyprinus carpio haematopterus) as raw material, the four fusion proteins-paired box 7 (Pax7), myogenic differentiation antigen (MyoD), myogenin (MyoG) and myogenic regulatory factor 4 (MRF4) for muscle development with antigenic determinant of Yellow River carp were obtained by prokaryotic expression technique. The fusion proteins were purified and used as antigen to immunize the New Zealand rabbits, correspondingly, four Yellow River carp polyclonal antibodies from rabbits were obtained. The enzyme linked immunosorbent assay (ELISA) method was used to evaluate the titers of antiserum, and the antibodies were used to detect the protein expression levels of the Pax7, MyoD, MyoG and MRF4 in white muscle of Yellow River carp (n=3). The results showed that the molecular weight of the four fusion proteins were about 15.60,16.90,16.91 and 19.35 ku, and the titers of the antiserum were all up to 2.4×10⁶. And there were no significant differences in the immunofluorescence signal intensities of Pax7, MyoD, MyoG and MRF4 in the white muscle of Yellow River carp (P>0.05). In conclusion, the polyclonal antibodies of key factors for muscle development prepared in this study can provide a powerful tool for further quantitative analysis of the expression of key marker proteins for muscle development in Yellow River carp.

Key words： Yellow River carp (Cyprinus carpio haematopterus); key factors of muscle development; polyclonal antibodies

Cite this article

YUN Yinghao , SONG Dongying , HE Zijie , MI Jiali , WANG Luming , NIE Guoxing . Preparation and Detection of Polyclonal Antibodies of Key Factors for Muscle Development of Yellow River Carp (Cyprinus carpio haematopterus)[J]. Chinese Journal of Animal Nutrition, 2022 , 34(3) : 1931 -1941 . DOI: 10.3969/j.issn.1006-267x.2022.03.052

References

[1] JOHNSTON I A,BOWER N I,MACQUEEN D J.Growth and the regulation of myotomal muscle mass in teleost fish[J].Journal of Experimental Biology,2011,214(Pt 10):1617-1628.
[2] RESCAN P Y.Development of myofibres and associated connective tissues in fish axial muscle:recent insights and future perspectives[J].Differentiation,2019,106:35-41.
[3] JOHNSTON I A.Environment and plasticity of myogenesis in teleost fish[J].The Journal of Experimental Biology,2006,209(Pt 12):2249-2264.
[4] JOHNSTON I A,ALDERSON R,SANDHAM C,et al.Muscle fibre density in relation to the colour and texture of smoked Atlantic salmon (Salmo salar L.)[J].Aquaculture,2000,189(3/4):335-349.
[5] LIU Z J,WANG C,LIU X Q,et al.Shisa2 regulates the fusion of muscle progenitors[J].Stem Cell Research,2018,31:31-41.
[6] RELAIX F,ROCANCOURT D,MANSOURI A,et al.A Pax3/Pax7-dependent population of skeletal muscle progenitor cells[J].Nature,2005,435(7044):948-953.

[7] RELAIX F,MONTARRAS D,ZAFFRAN S,et al.Pax3 and Pax7 have distinct and overlapping functions in adult muscle progenitor cells[J].Journal of Cell Biology,2006,172(1):91-102.

[8] 冯军厂,常绪路,朱振祥,等.鲤细胞因子多克隆抗体的制备及检测[J].水产学报,2018,42(10):1615-1625. FENG J C,CHANG X L,ZHU Z X,et al.Preparation and detection of cytokines polyclonal antibodies of Cyprinus carpio[J].Journal of Fisheries of China,2018,42(10):1615-1625.(in Chinese)
[9] ROWE R W,GOLDSPINK G.Muscle fibre growth in five different muscles in both sexes of mice[J].Journal of Anatomy,1969,104(Pt 3):519-530.
[10] DHAWAN J,RANDO T A.Stem cells in postnatal myogenesis:molecular mechanisms of satellite cell quiescence,activation and replenishment[J].Trends in Cell Biology,2005,15(12):666-673.

[11] JOHNSTON I A.Muscle development and growth:potential implications for flesh quality in fish[J].Aquaculture,1999,177(1/2/3/4):99-115.
[12] 赵新艳,郭妍婷,陈俊贞,等.牛骨骼肌卫星细胞的分离鉴定和诱导分化[J].中国畜牧兽医,2020,47(10):3249-3258. ZHAO X Y,GUO Y T,CHEN J Z,et al.Isolation,identification and differentiation of bovine skeletal muscle satellite cells[J].China Animal Husbandry & Veterinary Medicine,2020,47(10):3249-3258.(in Chinese)
[13] 刘志坚,方翊,王云龙,等.原代咬肌肌卫星细胞的提取及鉴定[J].口腔医学研究,2014,30(1):30-33. LIU Z J,FANG X,WANG Y L,et al.Isolation and differentiation of primary muscle satellite cell from rat masseter[J].Journal of Oral Science Research,2014,30(1):30-33.(in Chinese)
[14] GOTENSPARRE S M,ANDERSSON E,WARGELIUS A,et al.Insight into the complex genetic network of tetraploid Atlantic salmon (Salmo salar L.):description of multiple novel Pax-7 splice variants[J].Gene,2006,373:8-15.
[15] BENTZINGER C F,WANG Y X,RUDNICKI M A.Building muscle:molecular regulation of myogenesis[J].Cold Spring Harbor Perspectives in Biology,2012,4(2):a008342.
[16] GVNTHER S,KIM J,KOSTIN S,et al.Myf5-positive satellite cells contribute to Pax7-dependent long-term maintenance of adult muscle stem cells[J].Cell Stem Cell,2013,13(5):590-601.

[17] VON MALTZAHN J,JONES A E,PARKS R J,et al.Pax7 is critical for the normal function of satellite cells in adult skeletal muscle[J].Proceedings of the National Academy of Sciences of the United States of America,2013,110(41):16474-16479.

[18] 单立莉.Myostatin、MyoD、Myogenin基因对金华猪和长白猪肉质影响的比较研究[D].硕士学位论文.延吉:延边大学,2009. SHAN L L.Study on expressions of Myostatin,MyoD,Myogenin gene in meat quality between Jinhua pig and Landrance[D].Master's Thesis.Yanji:Yanbian University,2009.(in Chinese)
[19] 李虹辉,许友卿,刘小燕,等.鳜鱼生肌调节因子基因(MRF4)的克隆及其在成鱼和胚胎中的表达[J].湖南农业大学学报(自然科学版),2013,39(6):631-635. LI H H,XU Y Q,LIU X Y,et al.Molecular cloning of myogenic regulatory factor 4(MRF4) gene and its expression in adult and embryonic mandarin fish (Siniperca chuatsi)[J].Journal of Hunan Agricultural University (Natural Sciences),2013,39(6):631-635.(in Chinese)
[20] 李虹辉.翘嘴鳜(Siniperca chuatsi)生肌调节因子MRF4和MYF5的克隆及其表达研究[D].硕士学位论文.南宁:广西大学,2014. LI H H.Studies on the expression of MRF4 and MYF5 genes cloned from mandarin fish (Siniperca chuatsi)[D].Master's Thesis.Nanning:Guangxi University,2014.(in Chinese)
[21] 闫潇,杨丽萍,郑文佳,等.鲤肠道小肽转运载体PepT1多克隆抗体的制备及其组织表达分析[J].中国水产科学,2016,23(3):513-521. YAN X,YANG L P,ZHENG W J,et al.Preparation of the antibody and tissue distribution of the peptide transporter PepT1 in Cyprinus carpio L.[J].Journal of Fishery Sciences of China,2016,23(3):513-521.(in Chinese)
[22] 聂国兴,王贝,闫潇,等.鲤肠道sglt1基因的表达与抗体制备[J].水产学报,2012,36(3):329-335. NIE G X,WANG B,YAN X,et al.Expression of sglt1 gene in Cyprinus carpio and preparation of its polyclonal antibody[J].Journal of Fisheries of China,2012,36(3):329-335.(in Chinese)

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