鸡球虫病7价多表位嵌合重组抗原ET seven真核质粒的构建、表达及其初步功能分析

doi:10.11843/j.issn.0366-6964.2024.11.031

Abstract

Abstract:

The aim of this study was to construct multi-epitope recombinant plasmids of Eimeria tenella EtAMA1, EtAMA2, EtMIC3, EtMIC4, EtMIC13, EtROP5, and EtSAG1 antigens and verify their expression in DF-1 cells and chickens through various assisted delivery methods, we employed pcDNA3.1(+) as the eukaryotic expression vector. The eukaryotic expression plasmid pcDNA3.1-ET Seven was constructed through bioinformatics analysis, obtaining seven epitopes and reverse transcriptional amplification of gene fragments of seven antigen epitope regions in E. tenella mRNA templates at different time points. The company synthesized pcDNA3.1(+) as the eukaryotic expression vector, and constructed the eukaryotic expression plasmid pcDNA3.1-ET seven. Subsequently, the constructed positive plasmid, pcDNA3.1-ET Seven, was transfected into DF-1 cells. RT-PCR and Western blot analysis was employed to detect the expression of the ET Seven recombinant gene in DF-1 cells. The cytokine levels of pcDNA3.1-ET seven in chickens were detected by calcium phosphate nanoparticles as adjuvant plasmid delivery adjuvant, and specific IgG antibody levels were detected by indirect ELISA. The eukaryotic expression plasmid pcDNA3.1-ET Seven was successfully constructed, and the specific expression of ET Seven was detected through Western blot analysis and RT-PCR. After the plasmid was immunized with calcium phosphate nano-adjuvant, the detected cytokines IL-2, IL-6, IL-10, IL-12, IFN-γ and CD-40 were all expressed at high levels seven days after three times of immunization, and the expression levels of CD-40 and IL-6 were the highest. The pcDNA3.1-ET Seven recombinant plasmid, when conjugated with calcium phosphate adjuvant, was administered intramuscularly, resulting in a positive detection of specific IgG antibodies through indirect ELISA. The results showed that the pcDNA3.1-ET seven recombinant plasmid was successfully constructed and had good immunogenicity in chickens. This finding provides novel insights and technical support for vaccine prevention and control of chicken coccidiosis.

Key words: Eimeria tenella, multi-epitope recombinant plasmids, eukaryotic expression, DNA vaccine

CLC Number:

Junli NI, Xinchao LIU, Dong SUN, Siyun FANG, Dingai WANG, Hanqin SHEN, Zhuanqiang YAN, Nanshan QI, Mingfei SUN, Youfang GU. Construction, Expression and Preliminary Functional Analysis of the Eukaryotic Plasmid ET Seven, a 7-valent Multiepitope Chimeric Recombinant Antigen of Chicken Coccidia[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(11): 5159-5172.

Figures/Tables 12

Table 1

Table 2

Table 3

Table 4

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Fig. 6

Fig. 7

Fig. 8

References 51

1	SIBLEY L D .Intracellular parasite invasion strategies[J].Science,2004,304(5668):248-253. doi: 10.1126/science.1094717
2	BLAKE D P , KNOX J , DEHAECK B , et al.Re-calculating the cost of coccidiosis in chickens[J].Vet Res,2020,51(1):115. doi: 10.1186/s13567-020-00837-2
3	ZHANG L F .Multi-epitope vaccines: a promising strategy against tumors and viral infections[J].Cell Mol Immunol,2018,15(2):182-184. doi: 10.1038/cmi.2017.92
4	BIBI S , ULLAH I , ZHU B D , et al.In silico analysis of epitope-based vaccine candidate against tuberculosis using reverse vaccinology[J].Sci Rep,2021,11(1):1249. doi: 10.1038/s41598-020-80899-6
5	FADAKA A O , SIBUYI N R S , MARTIN D R , et al.Immunoinformatics design of a novel epitope-based vaccine candidate against dengue virus[J].Sci Rep,2021,11(1):19707. doi: 10.1038/s41598-021-99227-7
6	TAHIR M J. Isolation and characterization of dense granules of Eimeria tenella sporozoites[D]. Guelph: The University of Guelph., 1998.
7	TONKIN M L , ROQUES M , LAMARQUE M H , et al.Host cell invasion by Apicomplexan parasites: insights from the co-structure of AMA1 with a RON2 peptide[J].Science,2011,333(6041):463-467. doi: 10.1126/science.1204988
8	VULLIEZ-LE NORMAND B , TONKIN M L , LAMARQUE M H , et al.Structural and functional insights into the malaria parasite moving junction complex[J].PLoS Pathog,2012,8(6):e1002755. doi: 10.1371/journal.ppat.1002755
9	BESTEIRO S , MICHELIN A , PONCET J , et al.Export of a Toxoplasma gondii rhoptry neck protein complex at the host cell membrane to form the moving junction during invasion[J].PLoS Pathog,2009,5(2):e1000309. doi: 10.1371/journal.ppat.1000309
10	PASTOR-FERNÁNDEZ I , KIM S , BILLINGTON K , et al.Development of cross-protective Eimeria-vectored vaccines based on apical membrane antigens[J].Int J Parasitol,2018,48(7):505-518. doi: 10.1016/j.ijpara.2018.01.003
11	OLAJIDE J S , QU Z G , YANG S L , et al.Eimeria proteins: order amidst disorder[J].Parasit Vectors,2022,15(1):38. doi: 10.1186/s13071-022-05159-0
12	TOMLEY F M , SOLDATI D S .Mix and match modules: structure and function of microneme proteins in Apicomplexan parasites[J].Trends Parasitol,2001,17(2):81-88. doi: 10.1016/S1471-4922(00)01761-X
13	LI W Y , WANG M Y , CHEN Y F , et al.EtMIC3 and its receptors BAG1 and ENDOUL are essential for site-specific invasion of Eimeria tenella in chickens[J].Vet Res,2020,51(1):90. doi: 10.1186/s13567-020-00809-6
14	WANG X Q , WU L L , GAO Y , et al.Evaluation of the protective effect of pVAX-EtMIC3-recombined plasmid against E. tenella in chicken[J].Parasitol Res,2017,116(3):1023-1028. doi: 10.1007/s00436-017-5383-4
15	ZHANG X S , ZHAO Y J , ZHANG Y , et al.Role of EtMIC4 EGF-like in regulating the apoptosis of Eimeria tenella host cells via the EGFR pathway[J].Poult Sci,2022,101(10):102075. doi: 10.1016/j.psj.2022.102075
16	FRIEDRICH N , SANTOS J M , LIU Y , et al.Members of a novel protein family containing microneme adhesive repeat domains act as sialic acid-binding lectins during host cell invasion by Apicomplexan parasites[J].J Biol Chem,2010,285(3):2064-2076. doi: 10.1074/jbc.M109.060988
17	BEHNKE M S , FENTRESS S J , MASHAYEKHI M , et al.The polymorphic pseudokinase ROP5 controls virulence in Toxoplasma gondii by regulating the active kinase ROP18[J].PLoS Pathog,2012,8(11):e1002992. doi: 10.1371/journal.ppat.1002992
18	REESE M L , SHAH N , BOOTHROYD J C .The Toxoplasma pseudokinase ROP5 is an allosteric inhibitor of the immunity-related GTPases[J].J Biol Chem,2014,289(40):27849-27858. doi: 10.1074/jbc.M114.567057
19	CHOW Y P , WAN K L , BLAKE D P , et al.Immunogenic Eimeria tenella glycosylphosphatidylinositol-anchored surface antigens (SAGs) induce inflammatory responses in avian macrophages[J].PLoS One,2011,6(9):e25233. doi: 10.1371/journal.pone.0025233
20	REID A J , BLAKE D P , ANSARI H R , et al.Genomic analysis of the causative agents of coccidiosis in domestic chickens[J].Genome Res,2014,24(10):1676-1685. doi: 10.1101/gr.168955.113
21	JAHN D , MATROS A , BAKULINA A Y , et al.Model structure of the immunodominant surface antigen of Eimeria tenella identified as a target for sporozoite-neutralizing monoclonal antibody[J].Parasitol Res,2009,105(3):655-668. doi: 10.1007/s00436-009-1437-6
22	LIN Y H , WANG X , HUANG X F , et al.Calcium phosphate nanoparticles as a new generation vaccine adjuvant[J].Expert Rev Vaccines,2017,16(9):895-906. doi: 10.1080/14760584.2017.1355733
23	FATOLLAHZADEH M , ESKANDARIAN A , DARANI H Y , et al.Evaluation of Th17 immune responses of recombinant DNA vaccine encoding GRA14 and ROP13 genes against Toxoplasma gondii in BALB/c mice[J].Infect Genet Evol,2021,96,105150. doi: 10.1016/j.meegid.2021.105150
24	MOREIRA R S , FILHO V B , CALOMENO N A , et al.EpiBuilder: a tool for assembling, searching, and classifying B-Cell epitopes[J].Bioinform Biol Insights,2022,16,11779322221095221. doi: 10.1177/11779322221095221
25	QIU C , WU Y Y , GUO Q Y , et al.Preparation and application of calcium phosphate nanocarriers in drug delivery[J].Mater Today Bio,2022,17,100501. doi: 10.1016/j.mtbio.2022.100501
26	RABIEE N , BAGHERZADEH M , GHADIRI A M , et al.Calcium-based nanomaterials and their interrelation with chitosan: optimization for pCRISPR delivery[J].J Nanostruct Chem,2022,12(5):919-932. doi: 10.1007/s40097-021-00446-1
27	SOKOLOVA V V , RADTKE I , HEUMANN R , et al.Effective transfection of cells with multi-shell calcium phosphate-DNA nanoparticles[J].Biomaterials,2006,27(16):3147-3153. doi: 10.1016/j.biomaterials.2005.12.030
28	PEDRAZA C E , BASSETT D C , MCKEE M D , et al.The importance of particle size and DNA condensation salt for calcium phosphate nanoparticle transfection[J].Biomaterials,2008,29(23):3384-3392. doi: 10.1016/j.biomaterials.2008.04.043
29	GAGHAN C , ADAMS D , MOHAMMED J , et al.Characterization of vaccine-induced immune responses against coccidiosis in broiler chickens[J].Vaccine,2022,40(28):3893-3902. doi: 10.1016/j.vaccine.2022.05.043
30	黄诗雯, 宋硕, 王致萍, 等.人乳头瘤病毒治疗性疫苗的研究进展[J].病毒学报,2020,36(2):314-321.
	HUANG S W , SONG S , WANG Z P , et al.Research progress in human papillomavirus therapeutic vaccines[J].Chinese Journal of Virology,2020,36(2):314-321.
31	PASTON S J , BRENTVILLE V A , SYMONDS P , et al.Cancer vaccines, adjuvants, and delivery systems[J].Front Immunol,2021,12,627932. doi: 10.3389/fimmu.2021.627932
32	MITRA S , LEONARD W J .Biology of IL-2 and its therapeutic modulation: mechanisms and strategies[J].J Leukoc Biol,2018,103(4):643-655. doi: 10.1002/JLB.2RI0717-278R
33	TANAKA T , NARAZAKI M , KISHIMOTO T .IL-6 in inflammation, immunity, and disease[J].Cold Spring Harb Perspect Biol,2014,6(10):a016295. doi: 10.1101/cshperspect.a016295
34	KAUR S , BANSAL Y , KUMAR R , et al.A panoramic review of IL-6:structure, pathophysiological roles and inhibitors[J].Bioorg Med Chem,2020,28(5):115327. doi: 10.1016/j.bmc.2020.115327
35	SARAIVA M , VIEIRA P , O'GARRA A .Biology and therapeutic potential of interleukin-10[J].J Exp Med,2020,217(1):e20190418. doi: 10.1084/jem.20190418
36	HUTCHINS A P , DIEZ D , MIRANDA-SAAVEDRA D .The IL-10/STAT3-mediated anti-inflammatory response: recent developments and future challenges[J].Brief Funct Genomics,2013,12(6):489-498. doi: 10.1093/bfgp/elt028
37	BRAUN D A , FRIBOURG M , SEALFON S C .Cytokine response is determined by duration of receptor and signal transducers and activators of transcription 3 (STAT3) activation[J].J Biol Chem,2013,288(5):2986-2993. doi: 10.1074/jbc.M112.386573
38	VERSTOCKT B , SALAS A , SANDS B E , et al.IL-12 and IL-23 pathway inhibition in inflammatory bowel disease[J].Nat Rev Gastroenterol Hepatol,2023,20(7):433-446. doi: 10.1038/s41575-023-00768-1
39	TAIT WOJNO E D , HUNTER C A , STUMHOFER J S .The immunobiology of the interleukin-12 family: room for discovery[J].Immunity,2019,50(4):851-870. doi: 10.1016/j.immuni.2019.03.011
40	KELCHTERMANS H , BILLIAU A , MATTHYS P .How interferon-γ keeps autoimmune diseases in check[J].Trends Immunol,2008,29(10):479-486. doi: 10.1016/j.it.2008.07.002
41	GARRIS C S , ARLAUCKAS S P , KOHLER R H , et al.Successful anti-PD-1 cancer immunotherapy requires T cell-dendritic cell crosstalk involving the cytokines IFN-γ and IL-12[J].Immunity,2018,49(6):1148-1161. e7. doi: 10.1016/j.immuni.2018.09.024
42	PAN J P , ZHANG M H , WANG J L , et al.Interferon-gamma is an autocrine mediator for dendritic cell maturation[J].Immunol Lett,2004,94(1-2):141-151. doi: 10.1016/j.imlet.2004.05.003
43	CROFT M , BENEDICT C A , WARE C F .Clinical targeting of the TNF and TNFR superfamilies[J].Nat Rev Drug Discov,2013,12(2):147-168. doi: 10.1038/nrd3930
44	SHIMIZU K , ASAKURA M , FUJⅡ S I .Prolonged antitumor NK cell reactivity elicited by CXCL10-expressing dendritic cells licensed by CD40L⁺ CD4⁺ memory T cells[J].J Immunol,,2011,186(10):5927-5937. doi: 10.4049/jimmunol.1003351
45	ABELEV B , ADAM J , ADAMOVÁ D , et al.Exclusive J/ψ photoproduction off protons in ultraperipheral p-Pb collisions at $\sqrt{{ }^S \mathrm{NN}} $ =5. 02 TeV[J].Phys Rev Lett,2014,113(23):232504. doi: 10.1103/PhysRevLett.113.232504
46	XING L , FAN Y T , ZHOU T J , et al.Chemical modification of chitosan for efficient vaccine delivery[J].Molecules,,2018,23(2):229. doi: 10.3390/molecules23020229
47	SEFERIAN P G , MARTINEZ M L .Immune stimulating activity of two new chitosan containing adjuvant formulations[J].Vaccine,2000,19(6):661-668. doi: 10.1016/S0264-410X(00)00248-6
48	CARROLL E C , JIN L , MORI A , et al.The vaccine adjuvant chitosan promotes cellular immunity via DNA sensor cGAS-STING-dependent induction of type I interferons[J].Immunity,2016,44(3):597-608. doi: 10.1016/j.immuni.2016.02.004
49	SMITH T R F , PATEL A , RAMOS S , et al.Immunogenicity of a DNA vaccine candidate for COVID-19[J].Nat Commun,2020,11(1):2601. doi: 10.1038/s41467-020-16505-0
50	SONG K D , LILLEHOJ H S , CHOI K D , et al.A DNA vaccine encoding a conserved Eimeria protein induces protective immunity against live Eimeria acervulina challenge[J].Vaccine,2000,19(2-3):243-252. doi: 10.1016/S0264-410X(00)00169-9
51	DING X , LILLEHOJ H S , DALLOUL R A , et al.In ovo vaccination with the Eimeria tenella EtMIC2 gene induces protective immunity against coccidiosis[J].Vaccine,2005,23(28):3733-3740. doi: 10.1016/j.vaccine.2005.01.144

抗原片段 Antigen fragments	基因序列 Gene sequence	序列长度/ bp Sequence length
AMA1	CAAATAACCGTCCCCAACCCCACGGAGTGCGGCAAAGCTGTCTTCA- AAGTTTCTTCTTCAGACAACCCCACGCAGTACACGAAGCCACCCAC- CACTGAGGCCAGCAGCAGCACCAGCTCCAGCAACGCAGTGGCCACC- ATGTGGCCTGTGGGCGCCTTTTCCAAGGACGAACCCCGCATGCAGG- GGGTGGGCACCAACTATGCCAACTGGTACACCAATGGTACTTGTG- AGATGTATGACATGGTCCCCACTTGCTTCACCCTCGCCCCTAAC	273
AMA2	CCAGGGGGGACTTGTGTTATTTTTGCTGCTGTACCCTCTTGCTTCCT- GAGGGCCCCCGGGCACACAGCCTACACCTCAGTGGGTTCTTTGGCGG- AGGAGGCCCTCCCGGACTCCGCGCCGGGGGGCCCCCCCTCTTCTGGG- GGCGGGGGGGGCCCCCAGGAGCCCCAGGGGCCCCAGGAGCCCCAGCC- GGGGGGCCCCGAACCCGAACCCAGCCCCGAACCCAGCCCCGAACCCG- GAGAGCCCGGCAGCGGCGAAGGAGGGGGGGAAGAGGGGGAAACAA- ATCCTCCAAAAGAGCCTCAAGAAGAGCCTTCCTTGCCGCCTGAAGA- GGGGGGAGGTCAAGAAGAA	345
MIC3	GAACAGGGAAAAATAGGAAACTGGTGTTCCCAGAGTTGGGTTTTTT- GCTCAACTGCCATCATCTACGACCCCGCCTACTCCTACGGCACCTGCA- AGTGCGAGTCGTACAGAAACCGCTGCCCCATAGGCAGCAGCTGCCTC- GACTCCAGCTTCGGGGCTTTTTGCAAATGCAATGAAGACTTAGAAG- AGCGAAACGGGGGCTGCCACTTCACCACAACGACCACAACCACCACC- ACCACCACCACCACCACAACCACCACCACCACAACAACCACCACCACC- ACGCCCCCCGCGCGATCTCTCTGCAAA	309
MIC4	TTGCTCAAGGGCAAGAGCCTTGAGGATTGCAGAAAGGCTTGCTCAG- CAGACCAGAAGTGCACCAACTTCACGAGCTGGCTAAATAAGGAGTG- CTATATCAAGGATGACCAGGACCACCGGATGCAGGAACCAATACCG- GAGGCTACATCCGGATGGGTTTCGTGCTCAACG	171
MIC13	GGCGCCCTCGGAGTCTGGCGCTGCTACGCAGGGGCCACCATCAACCTC- AGTGCCCACAGCCTAGCTTGCGTCACCAACTGTGGGGACCCCATGAA- CTGCTCCGGACAGCTCACCCACATCTCTCCAATCTATTTCGATAGCA- ACTCCGACATGGTGGCAATCATCAAGCAGGAAAAGGTCAGGTACTG- CGGAAACTCGCGCTCTCGGCTGTCCACC	216
ROP5	CTAACGATTCCGTCGACCCCTCAAGTTGACATGAGTGGAAAAAGCC- CCATCGTCGTGCCACCTGTTCCAGCCGTTTTAGTCACTCCTGGGTCC- ACACGTATTGCGGTTGAGAGCCGAGAGACGGAAAGTGCAGGACAT- GGCACAGCAAGTGGAGTAAGCGCCACTGCCGGCGATGCTGCCAGGA- TCTCAGCAGGTACTAGC	201
SAG1	GACAGGGCTGTTTCCTTTGTCGCCCTATACAACCCCAAAACCAGCCC- CGTTGTCAGTTGCGTGCTCCTCCAGTGCCCTAATGCA	84

引物名称 Primer name	引物序列(5′→3′) Primer sequence	片段大小/bp Fragment length	退火温度/℃ Annealing temperature
AMA1-F	CAAATAACCGTCCCCAACC	273	54
AMA1-R	GTTAGGGGCGAGGGTGA		56
AMA2-F	CCAGGGGGGACTTGTGTTAT	345	57
AMA2-R	TTCTTCTTGACCTCCCCCC		56
MIC3-F	GAACAGGGAAAAATAGGAAACT	348	51
MIC3-R	ATCAATAACATCGCAGAAGCC		54
MIC4-F	GGCAGCGGAGAGAGCAAG	285	59
MIC4-R	CGTTGAGCACGAAACCCAT		57
MIC13-F	AATGATTCTCTGTGCCAAAACC	361	55
MIC13-R	ACTGCTGTTGCCCTTCCAT		58
ROP5-F	AACAGTGGTAAAATACACAAGGAC	501	54
ROP5-R	TTTTGTGCTCCTTGAGGTGCT		58
SAG1-F	ATGCCCCAAAGTCTTAGGAG	401	55
SAG1-R	TTTTCTTCCATTGCTCGTCGT		56

分组 Group	处理方式(肌肉多点位注射) Treatment (intramuscular multi-point injection)
空载体质粒组 Empty plasmid group	100 μg·羽^-1 pcDNA3.1质粒+磷酸钙纳米颗粒+壳聚糖
磷酸钙纳米颗粒佐剂对照组 Calcium phosphate nanoparticles adjuvant control group	磷酸钙纳米颗粒+壳聚糖(每羽剂量与免疫组相同)
空白对照组 Blank control group	不做任何处理
磷酸钙纳米颗粒免疫质粒组 Calcium phosphate nanoparticles immune plasmid group	100 μg·羽^-1 pcDNA3.1-ET seven质粒+磷酸钙纳米颗粒+壳聚糖

引物名称 Primer name	引物序列(5′→3′) Primer sequences	退火温度/℃ Annealing temperature
IL-2-F	TTGGAAAATATCAAGAACAAGATTCATC	50.9
IL-2-R	TCCCAGGTAACACTGCAGAGTTT	58.3
IL-6-F	CGTGTGCGAACAGCATGGAGA	60.9
IL-6-R	TCAGGCATTTCTCCTCGTCGAAGC	61.7
IL-10-F	AGCAGATCAAGGAGACGTTC	54.4
IL-10-R	ATCAGCAGGTACTCCTCGAT	55
IL-12-F	CCAAGACCTGGAGCACACACCGAAG	64.8
IL-12-R	CGATCCCTGGCCTGCACAGAGA	64.7
IFN-γ-F	ACACTGACAAGTCAAAGCCGCACA	61.7
IFN-γ-R	AGTCGTTCATCGGGAGCTTGGC	62.7
CD-40-F	CCTGGTGATGCTGTGAATTG	54.1
CD-40-R	CTTCTGTGTCGTTGCATTCAG	54.1
β-actin-F	CAACACAGTGCTGTCTGGTGGTA	59.6
β-actin-R	ATCGTACTCCTGCTTGCTGATCC	59.2

[1]	WANG Lele, WANG Liyue, CAI Weimin, KANG Xilong, FENG Qianqian, ZHANG Zhizhi, FAN Xuelian, ZHU Yu, LIU Dandan, XU Jinjun, PAN Zhiming, TAO Jianping. Immunoprotective Efficacy of Recombinant Protein rEnApiAP2 against Coccidia in Chicken [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(4): 1716-1727.
[2]	WANG He, GUO Zhiting, LI Jianxi, ZHANG Jingyan, WANG Lei, ZHANG Kang, SUN Jiwen, SHANG Xiaofen, MA Yonghua. Effect of Radix dichroa Powder on the Control of Eimeria tenella Infection in Chick at Different Developmental Stages [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(3): 1278-1289.
[3]	GAO Dengke, ZHAO Hongcong, DONG Hao, JIN Yaping, CHEN Huatao. The Cloning, Expression Vector Construction and Function Analysis of Goat RORα Gene [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(6): 1779-1794.
[4]	YUAN Jing, ZHANG Xinxin, ZHOU Di, YANG Shuai, ZHOU Jiayan, LIANG Aixin, YANG Liguo. Evaluation of the Effect of SS and CST Dual-expression DNA Vaccines with Attenuated Salmonella as Carrier in Immunizing Calves through Different Routes [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(6): 1914-1924.
[5]	JIAO Yuzhou, YANG Huanhuan, LU Yao, SHI Congcong, XU Xiaojuan, CAI Xuwang. Effect of Enterococcus faecium on Growth Performance and Intestinal Barrier of Broilers Infected with Eimeria tenella [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(4): 1210-1219.
[6]	LUO Ying, PENG Meiqi, XIAO Zhengpan, LUO Chenghui, WANG Dayong, PEI Yechun. Expression of Blomia tropicalis Major Allergens Blo t 5 and Blo t 21 Induced the Production of IgG and Regulatory T Cells in Mice [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(4): 1220-1230.
[7]	YIN Dejing, WU Yan, YUE Jun, YANG Peng, CHEN Jing, WANG Hui, ZHANG Shuangxiang, WANG Kaigong, CHENG Zhentao. Construction of Eukaryotic Expression Vector of N-terminal Gene of LppA Protein of Mycoplasma Mycoides subsp.Capri and Analysis of Its Immune Efficacy in Mice [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(3): 847-856.
[8]	LI Yajun, RU Yi, HAO Rongzeng, JIANG Chenghui, WANG Wei, ZHANG Yue, ZHANG Guicai, LIU Huanan, LU Bingzhou, YANG Yang, TAO Shiyu, YANG Rui, SONG Xiangdong, CHEN Jiao, YU Sijiu, ZHENG Haixue. Expression of Bovine Viral Diarrhea Virus E2 Protein in CHO Cells and Immunogenicity Analysis [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(12): 4315-4324.
[9]	ZHENG Tengfei, XIN Xiang, HAN Gaolian, LI Mengxin, LI Junling, QIN Jian, DU Rong. Construction of Eukaryotic Expression Vector of Sheep Musclin Gene and Its Effects on Glucose Metabolism and Insulin Function in Myocytes [J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(3): 641-652.
[10]	LI Xian, ZHANG Zhongwang, ZHANG Fudong, Lü Jianliang, LI Jiahao, PAN Li. Evaluation of the Characteristics of Mannosylated Chitosan PLGA Nanospheres as a Delivery System for DNA Vaccine of A/FMDV [J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(12): 3557-3568.
[11]	HAN Gaolian, GUO Lirong, LI Mengxin, ZHENG Tengfei, LI Junling, QIN Jian, DU Rong. Eukaryotic Expression of Sheep c-Myc and EGFP Fusion Protein and Its Regulation on the Cell Proliferation [J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(5): 965-975.
[12]	LIANG Shanshan, ZHU Shunhai, ZHAO Qiping, HUANG Bing, DONG Hui, YU Yu, WANG Qingjie, YU Shuilan, WANG Haixia, HAN Hongyu. Preliminary Study on the Function of Elongation Factor 1α of Eimeria tenella [J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(12): 3111-3121.
[13]	YANG Wei, WEI Guan-dong, TANG De-yuan, ZENG Zhi-yong, HUANG Tao, WANG Bin, HU Ling-ling, LONG Dong-mei, HUANG Qiu-han, LIAO Xiao-kang, TIAN Hong-yu. Japanese Encephalitis Virus Virulent Strains and Attenuated Strains of Non-Structural Proteins NS1, NS2A and NS1-NS2A Induce Mouse CD4⁺ T Cell Responses [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(8): 1693-1700.
[14]	LI Jun-min, YONG Yan-hong, GONG Dong-liang, FENG Yuan, WEI Xuan, HE Yu-lin, JU Xiang-hong. The Establishment of a Cell Line Stably Expressing Porcine GM-CSF [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(5): 963-970.
[15]	SUN Cheng-juan, XU Hou-qiang, DUAN Zhi-qiang, CHEN Wei, ZHAO Jia-fu, ZHOU Di. Construction of Recombinant Eukaryotic Expression Vector of FoxO 4 Gene from Congjiang Xiang Pig and Its Expression in Subcutaneous Preadipocytes [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(12): 2268-2276.

Construction, Expression and Preliminary Functional Analysis of the Eukaryotic Plasmid ET Seven, a 7-valent Multiepitope Chimeric Recombinant Antigen of Chicken Coccidia

RichHTML

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 12

References 51

Related Articles 15

Recommended Articles

Metrics

Comments