中国农业科学 ›› 2022, Vol. 55 ›› Issue (16): 3256-3266.doi: 10.3864/j.issn.0578-1752.2022.16.015
• 畜牧·兽医 • 上一篇
张冯禧1,2(),肖琦2,朱家平1,2,尹力鸿1,2,赵霞玲1,2,严明帅1,徐晋花1,温立斌2,牛家强1(
),何孔旺2(
)
收稿日期:
2022-03-14
接受日期:
2022-05-17
出版日期:
2022-08-16
发布日期:
2022-08-11
通讯作者:
牛家强,何孔旺
作者简介:
张冯禧,Tel:17767735839;E-mail: 基金资助:
ZHANG FengXi1,2(),XIAO Qi2,ZHU JiaPing1,2,YIN LiHong1,2,ZHAO XiaLing1,2,YAN MingShuai1,XU JinHua1,WEN LiBin2,NIU JiaQiang1(
),HE KongWang2(
)
Received:
2022-03-14
Accepted:
2022-05-17
Online:
2022-08-16
Published:
2022-08-11
Contact:
JiaQiang NIU,KongWang HE
摘要:
【背景】非洲猪瘟(ASF)于2018年8月在中国首次出现,对养猪业造成了巨大危害,损失惨重。目前尚无安全有效的疫苗用来预防ASF,于是建立快速特异的检测方法对于防控ASF提供了有效的手段。【目的】制备非洲猪瘟病毒(ASFV)特异性单克隆抗体,建立ASF快速特异性的检测方法。为ASF的检测和防控提供借鉴技术手段。【方法】构建表达载体pET-28a-P30,通过原核表达系统获得ASFV P30重组蛋白,以纯化的P30蛋白为抗原免疫BALB/c小鼠,经过细胞融合和细胞亚克隆制备出ASFV P30蛋白特异性杂交瘤细胞株;对P30蛋白进行截短表达,利用Western Blot和间接酶联免疫吸附试验(iELISA)鉴定单克隆抗体所对应的抗原表位;并利用制备的单克隆抗体建立非洲猪瘟阻断ELISA抗体检测方法。【结果】通过双酶切和PCR验证,结果显示构建出重组载体pET-28a-P30,经测序其序列未发生突变;IPTG诱导后,P30重组蛋白主要表达在包涵体中,分子量约为33 kD。纯化的P30蛋白与弗氏佐剂1﹕1混合免疫小鼠,3次免疫后,小鼠血清效价达到1﹕102 400,说明表达的蛋白具有良好的免疫原性。经细胞融合和亚克隆,获得8株P30蛋白特异性杂交瘤细胞,Western Blot和间接免疫荧光试验(IFA)检测获得的8株单抗均具有良好的反应性。叠加试验显示8株单克隆抗体均针对相同的抗原位点;截短表达P30蛋白不同片段,选取制备的2-12B单克隆抗体与不同的截短P30蛋白反应,显示单克隆抗体的抗原表位区为187—194aa。利用2-12B单克隆抗体并经过条件优化,成功建立了ASF阻断ELISA抗体检测方法,检测了190份临床样品,并与商品化非洲猪瘟ELISA抗体检测试剂盒进行对比,两方法阳性符合率为90.91%,总符合率为96.32%。【结论】本研究成功获得ASFV P30蛋白,经过iELISA、Western Blot和IFA筛选出反应性良好的特异性单克隆抗体8株,抗原识别表位区为187—194aa。并利用制备的单克隆抗体建立了特异性高,敏感性好的ASFV阻断ELISA抗体检测方法,为ASF的检测及其防控提供了手段和支撑。
张冯禧,肖琦,朱家平,尹力鸿,赵霞玲,严明帅,徐晋花,温立斌,牛家强,何孔旺. 非洲猪瘟病毒P30蛋白单克隆抗体制备、鉴定及阻断ELISA方法的建立[J]. 中国农业科学, 2022, 55(16): 3256-3266.
ZHANG FengXi,XIAO Qi,ZHU JiaPing,YIN LiHong,ZHAO XiaLing,YAN MingShuai,XU JinHua,WEN LiBin,NIU JiaQiang,HE KongWang. Preparation and Identification of Monoclonal Antibodies to P30 Protein and Establishment of Blocking ELISA to Detecting Antibodies Against African Swine Fever Virus[J]. Scientia Agricultura Sinica, 2022, 55(16): 3256-3266.
表1
P30蛋白截短表达引物表"
引物名称 Primer | 引物序列(5′-3′) Primer sequence (5′-3′) | 片段大小 Fragment size (bp) | |
---|---|---|---|
1-100aa | F | CGGGATCCCGATGGATTTTATTTTAAATATATCCATG | 343 |
R | CCCAAGCTTGGGTTAATGATGATGATGATGATGAGATGCTGAGGATTCCGTC | ||
50-150aa | F | CGGGATCCCGATGGCTATAAAAACATTGCTTAG | 349 |
R | CCCAAGCTTGGGTTAATGATGATGATGATGATGTTGTTCAATATGTTGCACA | ||
101-195aa | F | CGGGATCCCGATGTCGGAGAACATTCATGAAAAAAATGA | 328 |
R | CCCAAGCTTGGGTTAATGATGATGATGATGATGTTTTTTTTTTAAAAGTTTAATAA |
表2
P30蛋白 50-194aa截短表达引物表"
引物名称 Primer | 引物序列(5′-3′) Primer sequence (5′- 3′) | 片段大小 Fragment size (bp) |
---|---|---|
50-156aaR | CCCAAGCTTGGGTTAATGATGATGATGATGATGATCAGGTGCCTTTCCAT | 367 |
50-162aaR | CCCAAGCTTGGGTTAATGATGATGATGATGATGTCTAATAACCTTGTTAA | 385 |
50-168aaR | CCCAAGCTTGGGTTAATGATGATGATGATGATGTTGAATAAAATTATGTG | 403 |
50-174aaR | CCCAAGCTTGGGTTAATGATGATGATGATGATGAGGGGTTCCATAAATGG | 421 |
50-180aaR | CCCAAGCTTGGGTTAATGATGATGATGATGATGTTTTTCTTCTTCCTTTA | 439 |
50-186aaR | CCCAAGCTTGGGTTAATGATGATGATGATGATGCATGAGTCTTACCACCT | 457 |
50-192aaR | CCCAAGCTTGGGTTAATGATGATGATGATGATGTTTTAAAAGTTTAATAA | 475 |
[1] |
WALCZAK M, ŻMUDZKI J, MAZUR-PANASIUK N, JUSZKIEWICZ M, WOŹNIAKOWSKI G. Analysis of the clinical course of experimental infection with highly pathogenic African swine fever strain, isolated from an outbreak in Poland. aspects related to the disease suspicion at the farm level. Pathogens (Basel, Switzerland), 2020, 9(3): 237. doi: 10.3390/pathogens9030237.
doi: 10.3390/pathogens9030237 |
[2] |
LUONG H Q, LAI H T, DO L D, HA B X, NGUYEN G V, VU H L. Differential antibody responses in sows and finishing pigs naturally infected with African swine fever virus under field conditions. Virus Research, 2022, 307: 198621. doi: 10.1016/j.virusres.2021.198621.
doi: 10.1016/j.virusres.2021.198621 |
[3] |
VLASOV M, IMATDINOV A, TITOV I, VASKOVIĆ N, LYSKA V, SEVSKIKH T, SYBGATULLOVA A, PIVOVA E, MORGUNOV S, BALYSHEV V. Characteristics of African swine fever virus isolated from domestic pigs and wild boars in the Russian federation and south Ossetia. Acta Veterinaria, 2020, 70(1): 58-70. doi: 10.2478/acve-2020- 0004.
doi: 10.2478/acve-2020- 0004 |
[4] |
BISIMWA P N, ISHARA L K, WASSO D S, BANTUZEKO F, TONUI R, BWIHANGANE A B. Detection and genetic characterization of African swine fever virus (ASFV) in clinically infected pigs in two districts in South Kivu Province, Democratic Republic Congo. Heliyon, 2021, 7(3): e06419. doi: 10.1016/j.heliyon.2021.e06419.
doi: 10.1016/j.heliyon.2021.e06419 |
[5] |
GE S Q, LI J M, FAN X X, LIU F X, LI L, WANG Q H, REN W J, BAO J Y, LIU C J, WANG H, LIU Y T, ZHANG Y Q, XU T G, WU X D, WANG Z L. Molecular characterization of African swine fever virus, China, 2018. Emerging Infectious Diseases, 2018, 24(11): 2131-2133. doi: 10.3201/eid2411.181274.
doi: 10.3201/eid2411.181274 |
[6] |
LOPERA-MADRID J, MEDINA-MAGÜES L G, GLADUE D P, BORCA M V, OSORIO J E. Optimization in the expression of ASFV proteins for the development of subunit vaccines using poxviruses as delivery vectors. Scientific Reports, 2021, 11: 23476. doi: 10.1038/ s41598-021-02949-x.
doi: 10.1038/ s41598-021-02949-x |
[7] |
LABROUSSAA F, MEHINAGIC K, CIPPA V, LINIGER M, AKARSU H, RUGGLI N, JORES J. In-yeast reconstruction of the African swine fever virus genome isolated from clinical samples. STAR Protocols, 2021, 2(3): 100803. doi: 10.1016/j.xpro.2021.100803.
doi: 10.1016/j.xpro.2021.100803 |
[8] |
DIXON L K, CHAPMAN D A G, NETHERTON C L, UPTON C. African swine fever virus replication and genomics. Virus Research, 2013, 173(1): 3-14. doi: 10.1016/j.virusres.2012.10.020.
doi: 10.1016/j.virusres.2012.10.020 |
[9] |
LI D, ZHANG J, YANG W P, LI P, RU Y, KANG W F, LI L L, RAN Y, ZHENG H X. African swine fever virus protein MGF-505-7R promotes virulence and pathogenesis by inhibiting JAK1- and JAK2-mediated signaling. The Journal of Biological Chemistry, 2021, 297(5): 101190. doi: 10.1016/j.jbc.2021.101190.
doi: 10.1016/j.jbc.2021.101190 |
[10] |
JU X H, LI F, LI J R, WU C Y, XIANG G T, ZHAO X M, NAN Y C, ZHAO D M, DING Q. Genome-wide transcriptomic analysis of highly virulent African swine fever virus infection reveals complex and unique virus host interaction. Veterinary Microbiology, 2021, 261: 109211. doi: 10.1016/j.vetmic.2021.109211.
doi: 10.1016/j.vetmic.2021.109211 |
[11] |
HERRERA L, BISA E P. In silico analysis of highly conserved cytotoxic T-cell epitopes in the structural proteins of African swine fever virus. Veterinary World, 2021, 14(10): 2625-2633. doi: 10.14202/ vetworld.2021.2625-2633.
doi: 10.14202/ vetworld.2021.2625-2633 |
[12] |
LIU K F, MENG Y M, CHAI Y, LI L J, SUN H, GAO G F, TAN S G, QI J X. Crystal structure of the African swine fever virus core shell protein p15. Biosafety and Health, 2021, 3(2): 116-123. doi: 10.1016/j.bsheal.2020.09.002.
doi: 10.1016/j. bsheal.2020.09.002 |
[13] |
ZHAO G H, LI T T, LIU X M, ZHANG T Q, ZHANG Z X, KANG L, SONG J, ZHOU S J, CHEN X, WANG X, LI J N, HUANG L, LI C Y, BU Z G, ZHENG J, WENG C J. African swine fever virus cysteine protease pS273R inhibits pyroptosis by noncanonically cleaving gasdermin D. Journal of Biological Chemistry, 2022, 298(1): 101480. doi: 10.1016/j.jbc.2021.101480.
doi: 10.1016/j.jbc.2021.101480 |
[14] |
JIA N, OU Y W, PEJSAK Z, ZHANG Y G, ZHANG J. Roles of African swine fever virus structural proteins in viral infection. Journal of Veterinary Research, 2017, 61(2): 135-143. doi: 10.1515/jvetres- 2017-0017.
doi: 10.1515/jvetres- 2017-0017 |
[15] |
GAO Q, YANG Y L, QUAN W P, ZHENG J C, LUO Y Z, WANG H, CHEN X N, HUANG Z, CHEN X J, XU R D, ZHANG G H, GONG L. The African swine fever virus with MGF360 and MGF505 deleted reduces the apoptosis of porcine alveolar macrophages by inhibiting the NF-κB signaling pathway and interleukin-1β. Vaccines, 2021, 9(11): 1371. doi: 10.3390/vaccines9111371.
doi: 10.3390/vaccines9111371 |
[16] |
YANG K D, HUANG Q T, WANG R Y, ZENG Y, CHENG M Y, XUE Y, SHI C W, YE L P, YANG W T, JIANG Y L, WANG J Z, HUANG H B, CAO X, YANG G L, WANG C F. African swine fever virus MGF505-11R inhibits type I interferon production by negatively regulating the cGAS-STING-mediated signaling pathway. Veterinary Microbiology, 2021, 263: 109265. doi: 10.1016/j.vetmic.2021.109265.
doi: 10.1016/j.vetmic.2021.109265 |
[17] |
MURGIA M V, MOGLER M, CERTOMA A, GREEN D, MONAGHAN P, WILLIAMS D T, ROWLAND R R R, GAUDREAULT N N. Evaluation of an African swine fever (ASF) vaccine strategy incorporating priming with an alphavirus-expressed antigen followed by boosting with attenuated ASF virus. Archives of Virology, 2019, 164(2): 359-370. doi: 10.1007/s00705-018-4071-8.
doi: 10.1007/s00705-018-4071-8 |
[18] |
AFONSO C L, ALCARAZ C, BRUN A, SUSSMAN M D, ONISK D V, ESCRIBANO J M, ROCK D L. Characterization of P30, a highly antigenic membrane and secreted protein of African Swine Fever Virus. Virology, 1992, 189(1): 368-373. doi: 10.1016/0042-6822(92) 90718-5.
doi: 10.1016/0042-6822(92) 90718-5 |
[19] |
WU P, LOWE A D, RODRÍGUEZ Y Y, MURGIA M V, DODD K A, ROWLAND R R, JIA W. Antigenic regions of African swine fever virus phosphoprotein P30. Transboundary and Emerging Diseases, 2020: 2020Mar7. doi: 10.1111/tbed.13533.
doi: 10.1111/tbed.13533 |
[20] |
IMATDINOV A R, KAZAKOVA A S, ŠEKLER M, MOROZOVA D Y, LYSKA V M, TITOV I A, SEVSKIKH T, SEREDA A D. Immunization of pigs with recombinant plasmids containing genes of ubiquitinated p30, p54 and CD2v proteins of African swine fever virus. Acta Veterinaria, 2020, 70(1): 92-109. doi: 10.2478/acve-2020-0007.
doi: 10.2478/acve-2020-0007 |
[21] |
PETROVAN V, YUAN F F, LI Y H, SHANG P C, MURGIA M V, MISRA S, ROWLAND R R R, FANG Y. Development and characterization of monoclonal antibodies against p30 protein of African swine fever virus. Virus Research, 2019, 269: 197632. doi: 10.1016/j.virusres.2019.05.010.
doi: 10.1016/j.virusres.2019.05.010 |
[22] |
GALINDO I, ALONSO C. African swine fever virus: a review. Viruses, 2017, 9(5): 103. doi: 10.3390/v9050103.
doi: 10.3390/v9050103 |
[23] |
GOGIN A, GERASIMOV V, MALOGOLOVKIN A, KOLBASOV D. African swine fever in the North Caucasus region and the Russian Federation in years 2007-2012. Virus Research, 2013, 173(1): 198-203. doi: 10.1016/j.virusres.2012.12.007.
doi: 10.1016/j.virusres.2012.12.007 |
[24] |
SUN E C, ZHANG Z J, WANG Z L, HE X J, ZHANG X F, WANG L L, WANG W Q, HUANG L Y, XI F, HUANGFU H Y, TSEGAY G, HUO H, SUN J H, TIAN Z J, XIA W, YU X W, LI F, LIU R Q, GUAN Y T, ZHAO D M, BU Z G. Emergence and prevalence of naturally occurring lower virulent African swine fever viruses in domestic pigs in China in 2020. Science China Life Sciences, 2021, 64(5): 752-765. doi: 10.1007/s11427-021-1904-4.
doi: 10.1007/s11427-021-1904-4 |
[25] |
KIVUMBI C C, YONA C, HAKIZIMANA J N, MISINZO G. An assessment of the epidemiology and socioeconomic impact of the 2019 African swine fever outbreak in Ngara district, western Tanzania. Veterinary and Animal Science, 2021, 14: 100198. doi: 10.1016/j.vas. 2021.100198.
doi: 10.1016/j.vas. 2021.100198 |
[26] |
LI C F, HE X L, YANG Y, GONG W X, HUANG K, ZHANG Y F, YANG Y, SUN X M, REN W J, ZHANG Q, WU X D, ZOU Z, JIN M L. Rapid and visual detection of African swine fever virus antibody by using fluorescent immunochromatography test strip. Talanta, 2020, 219: 121284. doi: 10.1016/j.talanta.2020.121284.
doi: 10.1016/j.talanta.2020.121284 |
[27] |
GALLAGHER J R, HARRIS A K. Cryo-EM cools down swine fever. Journal of Biological Chemistry, 2020, 295(1): 13-14. doi: 10.1074/jbc. H119.012169.
doi: 10.1074/jbc. H119.012169 |
[28] |
CHEN Y Q, CHEN X, HUANG Q, SHAO Z W, GAO Y Q, LI Y Y, YANG C, LIU H H, LI J X, WANG Q Y, MA J B, ZHANG Y Z, GU Y J, GAN J H. A unique DNA-binding mode of African swine fever virus AP endonuclease. Cell Discovery, 2020, 6: 13. doi: 10.1038/ s41421-020-0146-2.
doi: 10.1038/ s41421-020-0146-2 |
[29] |
CHEN W Y, ZHAO D M, HE X J, LIU R Q, WANG Z L, ZHANG X F, LI F, SHAN D, CHEN H F, ZHANG J W, WANG L L, WEN Z Y, WANG X J, GUAN Y T, LIU J X, BU Z G. A seven-gene-deleted African swine fever virus is safe and effective as a live attenuated vaccine in pigs. Science China Life Sciences, 2020, 63(5): 623-634. doi: 10.1007/s11427-020-1657-9.
doi: 10.1007/s11427-020-1657-9 |
[30] |
SANNA G, DEI GIUDICI S, BACCIU D, ANGIOI P P, GIAMMARIOLI M, DE MIA G M, OGGIANO A. Improved strategy for molecular characterization of African swine fever viruses from Sardinia, based on analysis of p30, CD2V and I73R/I329L variable regions. Transboundary and Emerging Diseases, 2017, 64(4): 1280-1286. doi: 10.1111/tbed.12504.
doi: 10.1111/tbed.12504 |
[31] |
DIXON L K, SÁNCHEZ-CORDÓN P J, GALINDO I, ALONSO C. Investigations of pro- and anti-apoptotic factors affecting African swine fever virus replication and pathogenesis. Viruses, 2017, 9(9): 241. doi: 10.3390/v9090241.
doi: 10.3390/v9090241 |
[32] |
杨莎莎, 苗雨润, 薄宗义, 王振忠, 谭凯, 鲍晨沂, 吴晓东, 陈鸿军, 郑龙三, 钱莺娟, 戴建君. 非洲猪瘟病毒p30单克隆抗体的制备和鉴定. 中国兽医科学, 2020, 50(8): 976-981. doi: 10.16656/j.issn.1673-4696.2020.0117.
doi: 10.16656/j.issn. 1673-4696.2020.0117 |
YANG S S, MIAO Y R, BO Z Y, WANG Z Z, TAN K, BAO C Y, WU X D, CHEN H J, ZHENG L S, QIAN Y J, DAI J J. Generation and characterization of monoclonal antibodies against p30 protein of African swine fever virus. Chinese Veterinary Science, 2020, 50(8): 976-981. doi: 10.16656/j.issn.1673-4696.2020.0117. (in Chinese)
doi: 10.16656/j.issn. 1673-4696.2020.0117 |
|
[33] |
BARDERAS M G, WIGDOROVITZ A, MERELO F, BEITIA F, ALONSO C, BORCA M V, ESCRIBANO J M. Serodiagnosis of African swine fever using the recombinant protein p30 expressed in insect larvae. Journal of Virological Methods, 2000, 89(1/2): 129-136. doi: 10.1016/s0166-0934(00)00209-3.
doi: 10.1016/s0166-0934(00)00209-3 |
[34] |
ZHANG G L, LIU W, GAO Z, YANG S C, ZHOU G Q, CHANG Y Y, MA Y Y, LIANG X X, SHAO J J, CHANG H Y. Antigenicity and immunogenicity of recombinant proteins comprising African swine fever virus proteins p30 and p54 fused to a cell-penetrating peptide. International Immunopharmacology, 2021, 101(Pt A): 108251. doi: 10.1016/j.intimp.2021.108251.
doi: 10. 1016/j.intimp.2021.108251 |
[35] | 储德文. 非洲猪瘟病毒p30重组蛋白的表达及单克隆抗体制备[D]. 扬州: 扬州大学, 2013. |
CHU D W.Expression and monoclonal antibody preparation of the P 30 recombinant protein of African swine fever virus[D]. Yangzhou: Yangzhou University, 2013. (in Chinese) | |
[36] | 吕璐, 钟秋萍, 于婉琪, 谢振华, 张萌, 刘英楠, 叶建强, 秦爱建, 扈荣良, 陈鸿军. 非洲猪瘟病毒P30蛋白单克隆抗体的制备及鉴定. 中国动物传染病学报2020-11-25:1-10[网络首发], |
LÜ L, ZHONG Q P, YU W Q, XIE Z H, ZHANG M, LIU Y N, YE J Q, QIN A J, HU R L, CHEN H J Immunological Characterization of Monoclonal Antibodies Against African Swine Fever Virus P30. Chinese Journal of Animal Infectious Diseases, 2020-11-25: 1-10. (in Chinese) | |
[37] |
ZHOU G J, SHI Z W, LUO J C, CAO L Y, YANG B, WAN Y, WANG L J, SONG R, MA Y, TIAN H, ZHENG H X. Preparation and epitope mapping of monoclonal antibodies against African swine fever virus P30 protein. Applied Microbiology and Biotechnology, 2022, 106(3): 1199-1210. doi: 10.1007/s00253-022-11784-7.
doi: 10.1007/s00253-022-11784-7 |
[38] |
ZHANG X Y, LIU X Y, WU X D, REN W J, ZOU Y L, XIA X L, SUN H C. A colloidal gold test strip assay for the detection of African swine fever virus based on two monoclonal antibodies against P30. Archives of Virology, 2021, 166(3): 871-879. doi: 10.1007/s00705- 020-04915-w.
doi: 10.1007/s00705- 020-04915-w |
[1] | 魏天,王成宇,王凤杰,李忠鹏,张芳毓,张守峰,扈荣良,吕礼良,王永志. 非洲猪瘟病毒p30蛋白单克隆抗体制备及线性抗原表位定位[J]. 中国农业科学, 2022, 55(15): 3062-3070. |
[2] | 张静远,缪发明,陈腾,李敏,扈荣良. 非洲猪瘟实时荧光RPA诊断方法建立及应用[J]. 中国农业科学, 2022, 55(1): 197-207. |
[3] | 梁雨欣,吴建祥,李小宇,张春雨,侯吉超,周雪平,王永志. 马铃薯Y病毒衣壳蛋白抗原表位分析及其快速ELISA检测方法的建立[J]. 中国农业科学, 2021, 54(6): 1154-1162. |
[4] | 王涛,韩玉,潘力,王冰,孙茂文,王翌,罗玉子,仇华吉,孙元. 针对非洲猪瘟病毒MGF360-13L基因的TaqMan荧光定量PCR的建立[J]. 中国农业科学, 2021, 54(5): 1073-1080. |
[5] | 李敏雪,李剑男,周红,肖宁,蔺辉星,马喆,范红结. 基于SodC单克隆抗体的胞内劳森菌IPMA抗原检测方法的建立及应用[J]. 中国农业科学, 2021, 54(20): 4478-4486. |
[6] | 胡骁飞,李青梅,姚静静,胡思宇,孙亚宁,邢云瑞,邓瑞广,张改平. 基于结构类似物的交叉反应性制备高灵敏度 玉米赤霉醇单克隆抗体[J]. 中国农业科学, 2020, 53(5): 1071-1080. |
[7] | 郭亚璐,马晓飞,史佳楠,张柳,张剑硕,黄腾,武鹏程,康昊翔,耿广荟,陈浩,魏健,窦世娟,李莉云,尹长城,刘国振. 转基因水稻中CAS9蛋白质的免疫印迹检测[J]. 中国农业科学, 2017, 50(19): 3631-3639. |
[8] | 谭永安,肖留斌,郝德君,赵静,孙洋,柏立新. 绿盲蝽AlEcR-A的单克隆抗体制备及在外源20E诱导下的应答[J]. 中国农业科学, 2017, 50(1): 86-93. |
[9] | 陈 浙,宋 革,周雪平,吴建祥. 西瓜花叶病毒(WMV)单克隆抗体的制备及其应用[J]. 中国农业科学, 2016, 49(14): 2711-2724. |
[10] | 赵丹丹,杨国平,刁有祥,陈 浩,提金凤,张 璐,张 英,李川川. 鸭瘟病毒单抗的制备及胶体金试纸条检测方法的建立[J]. 中国农业科学, 2016, 49(14): 2796-2804. |
[11] | 赵鑫,高美须,牟慧,沈月,王志东. 模拟胃液对Pena 1及其抗原表位免疫原性的影响[J]. 中国农业科学, 2015, 48(4): 769-777. |
[12] | 牟慧1, 高美须1, 潘家荣2, 支玉香3, 赵杰1, 刘超超1, 李树锦1, 赵鑫1. Pen a1抗原表位187—202关键氨基酸的筛选和鉴定[J]. 中国农业科学, 2014, 47(9): 1793-1801. |
[13] | 黄娇玲, 谢芝勋, 谢丽基, 滕丽琼, 黄莉, 刘加波, 庞耀珊, 邓显文, 谢志勤, 范晴, 罗思思. 基于纳米材料电化学免疫传感器检测禽呼肠孤病毒研究[J]. 中国农业科学, 2014, 47(5): 1006-1012. |
[14] | 李新朋,姜金庆,钱爱东,王自良,范国英,单晓峰,康元环,李艺. 氟喹诺酮类药物多残留酶联免疫检测方法的建立[J]. 中国农业科学, 2014, 47(23): 4726-2735. |
[15] | 王玲玲, 职爱民, 杨艳艳, 宋春美, 王坤, 柴书军, 侯玉泽, 邓瑞广, 张改平. 抗百菌清单克隆抗体的研制与鉴定[J]. 中国农业科学, 2013, 46(7): 1509-1515. |
|