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

doi:10.3864/j.issn.0578-1752.2022.16.015

Abstract

Abstract:

【Background】 African swine fever (ASF) first appeared in China in August 2018, causing great harm to the pig industry and heavy losses. At present, there is no safe and effective vaccine to prevent ASF, so the establishment of rapid and specific detection method provides an effective means for the prevention and control of ASF. 【Objective】To prepare specific monoclonal antibodies against African swine fever virus (ASFV) and establish a rapid and specific detection method for ASF. Provide technical means for the detection and prevention and control of ASF. 【Method】The expression vector pET-28a-P30 was constructed and the rP30 protein was obtained by prokaryotic expression system. The purified rP30 protein was used as antigen to immunize BALB/c mice. The specific hybridoma cell line of ASFV P30 protein was prepared by cell fusion and cell subcloning. The truncated expression of P30 protein was performed, and Western Blot and indirect enzyme-linked immunosorbent assay (iELISA) were used to identify the antigen epitope corresponding to the monoclonal antibody. The prepared monoclonal antibody was used to establish the detection method of blocking ELISA antibody for ASF. 【Result】 The results of double digestion and PCR showed that the recombinant vector pET-28a-P30 was constructed, and the sequence was not mutated by sequencing. After IPTG induction, the recombinant P30 protein was mainly expressed in the inclusion body with a molecular weight of about 33 kD. The purified P30 protein was mixed with 1﹕1 Freund's adjuvant to immunize mice. After three immunizations, the serum titer of mice reached 1﹕102 400, indicating that the expressed protein had good immunogenicity. Eight P30 protein-specific hybridoma cells were obtained by cell fusion and subcloning. The eight monoclonal antibodies obtained by Western Blot and indirect immunofluorescence assay (IFA) showed good reactivity. The superposition test showed that all the 8 monoclonal antibodies had the same antigenic sites. Different fragments of P30 protein were truncated, and the prepared 2-12B monoclonal antibodies were selected to react with different truncated P30 proteins, showing that the antigenic epitope region of the monoclonal antibodies was 187-194aa. The ASF blocking ELISA antibody detection method was successfully established by using 2-12B monoclonal antibody and optimizing the conditions. 190 clinical samples were detected and compared with commercial African swine fever ELISA antibody detection kit. The positive coincidence rate of the two methods was 90.91 %, and the total coincidence rate was 96.32 %. 【Conclusion】 In this study, ASFV P30 protein was successfully obtained. Eight specific monoclonal antibodies with good reactivity were screened by iELISA, Western Blot and IFA, and the antigen recognition epitopes were 187-194 aa. The monoclonal antibody was used to establish a high specificity and sensitivity ASFV blocking ELISA antibody detection method, which provided a means and support for the detection and prevention of ASF.

Key words: African swine fever virus, p30 protein, monoclonal antibody, antigen epitope, blocking ELISA

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.

0
/ / Recommend

Add to citation manager EndNote|Reference Manager|ProCite|BibTeX|RefWorks

URL: https://www.chinaagrisci.com/EN/10.3864/j.issn.0578-1752.2022.16.015

https://www.chinaagrisci.com/EN/Y2022/V55/I16/3256

Figures/Tables 13

Table 1

Table 2

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Fig. 6

Fig. 7

Fig. 8

Table 3

Table 4

Table 5

References 38

[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

Metrics

Viewed

Full text

Abstract

Cited

Shared

Discussed

Comments

Recommended 0

No Suggested Reading articles found!

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
1-100aa	R	CCCAAGCTTGGGTTAATGATGATGATGATGATGAGATGCTGAGGATTCCGTC	343
50-150aa	F	CGGGATCCCGATGGCTATAAAAACATTGCTTAG	349
50-150aa	R	CCCAAGCTTGGGTTAATGATGATGATGATGATGTTGTTCAATATGTTGCACA	349
101-195aa	F	CGGGATCCCGATGTCGGAGAACATTCATGAAAAAAATGA	328
101-195aa	R	CCCAAGCTTGGGTTAATGATGATGATGATGATGTTTTTTTTTTAAAAGTTTAATAA	328

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

阻断ELISA检测 Blocking ELISA detection	间接ELISA检测试剂盒 Indirect ELISA kit		合计 Total
阻断ELISA检测 Blocking ELISA detection	+	-	合计 Total
+	20	5	25
-	2	163	165
合计Total	22	168	190

Preparation and Identification of Monoclonal Antibodies to P30 Protein and Establishment of Blocking ELISA to Detecting Antibodies Against African Swine Fever Virus

RichHTML

PDF

Abstract

Cite this article

share this article

Figures/Tables 13

References 38

Related Articles 15

Metrics

Comments

Recommended 0

[1]	WEI Tian,WANG ChengYu,WANG FengJie,LI ZhongPeng,ZHANG FangYu,ZHANG ShouFeng,HU RongLiang,LÜ LiLiang,WANG YongZhi. Preparation of Monoclonal Antibodies Against the p30 Protein of African Swine Fever Virus and Its Mapping of Linear Epitopes [J]. Scientia Agricultura Sinica, 2022, 55(15): 3062-3070.
[2]	ZHANG JingYuan,MIAO FaMing,CHEN Teng,LI Min,HU RongLiang. Development and Application of a Real-Time Fluorescent RPA Diagnostic Assay for African Swine Fever [J]. Scientia Agricultura Sinica, 2022, 55(1): 197-207.
[3]	YuXin LIANG,JianXiang WU,XiaoYu LI,ChunYu ZHANG,JiChao HOU,XuePing ZHOU,YongZhi WANG. Mapping of Epitopes and Establishment of Rapid DAS-ELISA for Potato Virus Y Coat Protein [J]. Scientia Agricultura Sinica, 2021, 54(6): 1154-1162.
[4]	Tao WANG,Yu HAN,Li PAN,Bing WANG,MaoWen SUN,Yi WANG,YuZi LUO,HuaJi QIU,Yuan SUN. Development of a TaqMan Real-Time PCR Targeting the MGF360-13L Gene of African Swine Fever Virus [J]. Scientia Agricultura Sinica, 2021, 54(5): 1073-1080.
[5]	LI MinXue,LI JianNan,ZHOU Hong,XIAO Ning,LIN HuiXing,MA Zhe,FAN HongJie. Establishment and Preliminary Application of Lawsonia intracellularis IPMA Antigen Detection Method Based on SodC Monoclonal Antibody [J]. Scientia Agricultura Sinica, 2021, 54(20): 4478-4486.
[6]	HU XiaoFei,LI QingMei,YAO JingJing,HU SiYu,SUN YaNing,XING YunRui,DENG RuiGuang,ZHANG GaiPing. Development of High Sensitive Zeranol Monoclonal Antibody Based on the Cross Reactivity of Structural Analogs [J]. Scientia Agricultura Sinica, 2020, 53(5): 1071-1080.
[7]	GUO YaLu, MA XiaoFei, SHI JiaNan, ZHANG Liu, ZHANG JianShuo, HUANG Teng, WU PengCheng, KANG HaoXiang, GENG GuangHui, CHEN Hao, WEI Jian, DOU ShiJuan, LI LiYun, YIN ChangCheng, LIU GuoZhen . Western Blot Detection of CAS9 Protein in Transgenic Rice [J]. Scientia Agricultura Sinica, 2017, 50(19): 3631-3639.
[8]	CHEN Zhe, SONG Ge, ZHOU Xue-ping, WU Jian-xiang. Preparation and Application of Monoclonal Antibodies Against Watermelon mosaic virus (WMV) [J]. Scientia Agricultura Sinica, 2016, 49(14): 2711-2724.
[9]	ZHAO Dan-dan, YANG Guo-ping, DIAO You-xiang, CHEN Hao, TI Jin-feng, ZHANG Lu, ZHANG Ying, LI Chuan-chuan. Preparation of Monoclonal Antibodies Against DPV and Development of Colloidal Gold Strip for DPV Detection [J]. Scientia Agricultura Sinica, 2016, 49(14): 2796-2804.
[10]	WANG Ling-Ling, ZHI Ai-Min, YANG Yan-Yan, SONG Chun-Mei, WANG Kun, CHAI Shu-Jun, HOU Yu-Ze, DENG Rui-Guang, ZHANG Gai-Ping. Preparation and Identification of the Monoclonal Antibody Against Chlorothalonil [J]. Scientia Agricultura Sinica, 2013, 46(7): 1509-1515.
[11]	GONG Fang, ZHI Ai-Min, LI Qing-Mei, HU Xiao-Fei, ZHAO Qi-Fa, ZHANG Xiao-Hui, LU Qi-Yu, ZHANG Gai-Ping. Establishment of Hybridoma Cell Lines Secreting Anti-Kasugamycin Monoclonal Antibody and Identification of Their Immunological Properties [J]. Scientia Agricultura Sinica, 2013, 46(2): 417-423.
[12]	XING Guang-Xu-12, WANG Guo-Dong-3, HU Xiao-Fei-2, WANG Fang-Yu-2, DENG Rui-Guang-2, ZHANG Gai-Ping-2. Development and Identification of ELISA Kit for Norfloxacin Determination [J]. Scientia Agricultura Sinica, 2013, 46(16): 3470-3477.
[13]	^{WANG Ai-ping,LI Fa-di,HU Xiao-fei,SHI Zhao-guo,ZHOU Jing-ming,DU Xiao-ming,WANG Lin-lin,YU Le-le,LIU Ming-yang}. Development of an Immunochromatographic Strip Test for Detection of Neomycin [J]. Scientia Agricultura Sinica, 2011, 44(11): 2387-2397 .
[14]	ZHI Ai-min,LI Qing-mei,LIU Qing-tang,LIU Xuan-bing,YANG Ji-fei,YANF Su-zhen,CHAI Shu-jun,YANG Yan-yan,DENG Rui-guang,ZHANG Gai-ping . Preparation and Preliminary Application of Anti-Gentamicin Monoclonal Antibody #br# [J]. Scientia Agricultura Sinica, 2010, 43(12): 2584-2589 .
[15]	. Preparation of Monoclonal Antibody Against PthA-NLS and Construction of the Relative ScFv Gene [J]. Scientia Agricultura Sinica, 2009, 42(3): 884-890 .