鸡柔嫩艾美耳球虫2种假定致密颗粒蛋白基因的克隆与表达

doi:10.3969/j.issn.1004-1524.20230300

浙江农业学报 ›› 2024, Vol. 36 ›› Issue (3): 503-514.DOI: 10.3969/j.issn.1004-1524.20230300

鸡柔嫩艾美耳球虫2种假定致密颗粒蛋白基因的克隆与表达

李天恩¹^,²(), 周思含¹^,², 孙洪超², 付媛², 石团员²^,^*(), 闫文朝¹^,^*()

1.河南科技大学动物科技学院,河南洛阳 471023
2.浙江省农业科学院畜牧兽医研究所,浙江杭州 310021

收稿日期:2023-03-10 出版日期:2024-03-25 发布日期:2024-04-09
作者简介:李天恩(1996—),男,河南周口人,硕士研究生,研究方向为动物寄生虫病学。E-mail: litianen0509@163.com
通讯作者: *石团员,E-mail:lstone2008@126.com;闫文朝,E-mail:ywchao11@126.com
基金资助:
国家自然科学基金(31472182);杭州市农业与社会发展项目(202203B19);浙江省“三农九方”项目(2023SNJF058)

Cloning and expression analysis of two hypothetical dense granule protein genes of Eimeria tenella in chickens

LI Tian’en¹^,²(), ZHOU Sihan¹^,², SUN Hongchao², FU Yuan², SHI Tuanyuan²^,^*(), YAN Wenchao¹^,^*()

1. College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471023, Henan, China
2. Institute of Animal Husbandry and Veterinary Medicine, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China

Received:2023-03-10 Online:2024-03-25 Published:2024-04-09

摘要/Abstract

摘要：

柔嫩艾美耳球虫(Eimeria tenella)是一种严重危害养鸡业健康发展的高致病性原虫,致密颗粒蛋白(dense granule proteins, GRAs)是顶复合器门原虫胞内寄生重要功能蛋白,具有较高的免疫学应用价值,然而目前尚未有关于球虫致密颗粒蛋白的确切报道。为发掘柔嫩艾美耳球虫GRAs并研究其功能,采用生物信息学、分子生物学、免疫学等技术从E. tenella北京株中鉴定到2种假定致密颗粒蛋白(hypothetical dense granule protein, hEtGRA)hEtGRA12、hEtGRA9,并用纯化后的蛋白分别免疫小鼠制备多克隆抗体,用ELISA、Western blot方法检测2种蛋白的抗原性。分子克隆结果表明,hEtGRA12、hEtGRA9基因编码区长度分别为1 188、1 110 bp,分别编码395、369个氨基酸,与其他顶复合器门原虫致密颗粒蛋白GRA12、GRA9物种间相似性分别为28.8%~39.6%、27.5%~29.5%;SDS-PAGE结果显示,重组蛋白rhEtGRA12、rhEtGRA9条带大小分别为63.6、67.0 ku;ELISA与Western blot结果表明,重组蛋白rhEtGRA12、rhEtGRA9均能诱导小鼠产生较高的抗体水平,且均可被鸡抗E. tenella阳性血清识别,表明具有较好的抗原性。该研究鉴定到2种球虫假定致密颗粒蛋白基因hEtGRA12、hEtGRA9,获得了重组蛋白rhEtGRA12、rhEtGRA9,为球虫致密颗粒蛋白基因功能和免疫应用研究奠定了基础。

关键词: 柔嫩艾美耳球虫, 假定致密颗粒蛋白, 原核表达, 多克隆抗体, 抗原性

Abstract:

Eimeria tenella is a highly pathogenic protozoan that seriously endangers the healthy development of the chicken industry. Dense granule proteins (GRAs) are important intracellular parasitic functional proteins of the apicomplexan protozoa, which have high immunological application value. However, there is no exact report on the dense granule proteins of coccidia. In order to explore the GRAs of Eimeria tenella and study their functions, two hypothetical dense granule proteins (hEtGRA), hEtGRA12 and hEtGRA9, were identified from E. tenella Beijing strain by bioinformatics, molecular biology and immunology. The purified proteins were used to immunize mice to prepare polyclonal antibodies, and the antigenicity of the two proteins was detected by ELISA and Western blot. The results of molecular cloning showed that the coding regions of hEtGRA12 and hEtGRA9 genes were 1 188 bp and 1 110 bp in length, encoding 395 and 369 amino acids, respectively. The similarity of hEtGRA12 and hEtGRA9 with GRA12 and GRA9 of other apicomplexan protozoa was 28.8%-39.6% and 27.5%-29.5%, respectively. SDS-PAGE showed that the recombinant proteins rhEtGRA12 and rhEtGRA9 were 63.6 ku and 67.0 ku, respectively. The results of ELISA and Western blot showed that the recombinant proteins rhEtGRA12 and rhEtGRA9 could induce high antibody levels in mice, and could be recognized by chicken anti-E. tenella positive serum, indicating good antigenicity. In this study, two hypothetical dense granule protein genes hEtGRA12 and hEtGRA9 of Eimeria were identified, and recombinant proteins rhEtGRA12 and rhEtGRA9 were obtained, which laid a foundation for the study of gene function and immune application of dense granule protein of Eimeria.

Key words: Eimeria tenella, hypothetical dense granule protein, prokaryotic expression, polyclonal antibody, antigenicity

中图分类号:

S858.31

李天恩, 周思含, 孙洪超, 付媛, 石团员, 闫文朝. 鸡柔嫩艾美耳球虫2种假定致密颗粒蛋白基因的克隆与表达[J]. 浙江农业学报, 2024, 36(3): 503-514.

LI Tian’en, ZHOU Sihan, SUN Hongchao, FU Yuan, SHI Tuanyuan, YAN Wenchao. Cloning and expression analysis of two hypothetical dense granule protein genes of Eimeria tenella in chickens[J]. Acta Agriculturae Zhejiangensis, 2024, 36(3): 503-514.

图/表 9

表1 克隆E. tenella假定致密颗粒蛋白基因序列所用引物

Table 1 Primers for cloning dense granule protein hypothetical genes in E. tenella

假定致密颗粒蛋白基因 Hypothetical dense granule protein gene	上游引物序列 Forward primer sequences(5'→3')	下游引物序列 Reverse primer sequences(5'→3')
ETH_00023950	ATGGGATTGACTGGCTTTT	TCATGAGTCTCCCTTACTC
ETH_00031740	ATGGCGCCCCGGCTTTCC	CTAGGCCGCCTCCTCCTCGTTGTCC
ETH_00024035	ATGCGATCCTCGCAGCGT	TTAATTTCTCTTCGAGGCCG
ETH_00028475	ATGCCCCACCACGTGCTGG	TCAGCAGAGAAAGGCTGCATAGC
ETH_00009660	ATGGTATATCTGTTATTGCC	CTAGCTAGCCAAGTAGGCACTTGCC
ETH_00025090	ATGCCTGCAGCTGCAGCTCCC	TCAAAACTGCATCTCCAGCAGCAGC
ETH_00023930	ATGGTTGTGTCGTTGATTGTGTGTG	TTAACTTTGCGCCCTCTCTGCTTCA
ETH_00035235	ATGAATGAAGTCGAAGATGTCC	TCAGCACAGCAGGCCGCAGCCCACC
ETH_00030725	ATGCCCTTAAAGATGGCTTTGC	TTAGTCATTTGTTGCTGTAGCAACA
ETH_00010075	ATGCGGCGTCTGCAGCTGAACT	TGTTGCTGCTTCTGCTGCTGCTGCT
ETH_00032355	ATGAAAATGAAAAAAGAAAATC	TCATTCATCTCCAATCCACATCCGC

图1 hEtGRA12、hEtGRA9基因的克隆 M,DNA marker;1,hEtGRA12基因片段;2,hEtGRA9基因片段。

Fig.1 Cloning of hEtGRA12 gene and hEtGRA9 gene M, DNA maker; 1, hEtGRA12 gene fragment; 2, hEtGRA9 gene fragment.

图2 hEtGRA12蛋白同源性与系统发育分析 A,同源性分析;B,序列相似性分析;C,亲缘关系分析,△表示hEtGRA12蛋白序列。

Fig.2 Homology and phylogenetic analysis of hEtGRA12 protein A, Analysis of homolog; B, Analysis of sequence similarity; C, Analysis of kinship, △ represented sequence of hEtGRA12 protein.

图3 hEtGRA9蛋白序列相似性分析 A,hEtGRA9与TgGRA9蛋白序列相似性分析;B,hEtGRA9与BbGRA9蛋白序列相似性分析。●,hEtGRA9蛋白序列;▲,TgGRA9蛋白序列;◆,BbGRA9蛋白序列。

Fig.3 Sequence similarity analysis of hEtGRA9 protein A, Sequence similarity analysis of hEtGRA9 and TgGRA9 protein; B, Sequence similarity analysis of hEtGRA9 and BbGRA9 protein. ●,Sequence of hEtGRA9 protein; ▲, Sequence of TgGRA9 protein; ◆, Sequence of BbGRA9 protein.

图4 hEtGRA12蛋白的生物信息学分析 A,信号肽预测;B,跨膜区预测;C,亲/疏水性分析;D,二级结构预测,蓝色代表α螺旋,紫色代表无规则卷曲,红色代表延伸链;E,抗原表位预测。图5同。

Fig.4 Bioinformatics analysis of EtGRA12 protein A, Prediction of signal peptide; B, Prediction of transmembrane region; C, Analysis of hydrophilic/hydrophobic; D, Prediction of secondary structure, blue represented α-helix, purple represented random coi, red represented extended strand; E, Prediction of antigen epitope. The same as in figure 5.

图5 hEtGRA9蛋白的生物信息学分析

Fig.5 Bioinformatics analysis of hEtGRA9 protein

图6 蛋白功能结构域保守性分析绿色,信号肽;蓝色,α螺旋;紫色,无规则卷曲;红色,延伸链。

Fig.6 Conservative analysis of protein functional domain Green, Signal peptide; Blue, α-Helix; Purple, Random coil; Red, Extended strand.

图7 hEtGRA12、hEtGRA9基因的重组表达 A,pET32a(+)-hEtGRA12质粒双酶切鉴定;B,hEtGRA12蛋白的表达纯化;C,pET32a(+)-hEtGRA9质粒双酶切鉴定;D,hEtGRA9蛋白的表达纯化。M1,DNA marker;M2,蛋白分子量标准;1,pET32a(+)-hEtGRA12质粒;2,pET32a(+)-hEtGRA12质粒双酶切产物;3,未纯化的hEtGRA12蛋白;4~5,纯化的hEtGRA12蛋白;6,pET32a(+)-hEtGRA9质粒;7,pET32a(+)-hEtGRA9质粒双酶切产物;8,未纯化的hEtGRA9蛋白;9,纯化的hEtGRA9蛋白。

Fig.7 Recombinant expression of hEtGRA12 and hEtGRA9 genes A, Identification of pET32a (+)-hEtGRA12 plasmid by double enzyme digestion; B, Expression and purification of hEtGRA12 protein; C, Identification of pET32a (+)-hEtGRA9 plasmid by double enzyme digestion; D, Expression and purification of hEtGRA9 protein; M1, DNA maker; M2, Protein maker; 1, pET32a (+)-hEtGRA12 plasmid; 2, Digestion product of pET32a(+)-hEtGRA12 plasmid; 3, Unpurified hEtGRA12 protein; 4-5, Purified hEtGRA12 protein; 6, pET32a(+)-hEtGRA9 plasmid; 7, Digestion product of pET32a(+)-hEtGRA9 plasmid; 8, Unpurified hEtGRA9 protein; 9, Purified hEtGRA9 protein.

图8 rhEtGRA12、rhEtGRA9抗原性分析 A,重组蛋白rhEtGRA12 Western blot分析;B,重组蛋白rhEtGRA9 Western blot分析;M,蛋白质分子量标准;1、4,阴性鸡血清;2、3,抗柔嫩艾美耳球虫阳性鸡血清;C,鼠抗rhEtGRA12、鼠抗rhEtGRA9多克隆抗体效价检测。

Fig.8 Antigenicity analysis of rhEtGRA12 and rhEtGRA9 A, Western blot analysis of recombinant protein rhEtGRA12; B, Western blot analysis of recombinant protein rhEtGRA9; M, Protein marker; 1 and 4, Negative chicken serum; 2 and 3, Anti-Eimeria tenella positive chicken serum; C, Detection of mouse anti-rhEtGRA12 and mouse anti-rhEtGRA9 polyclonal antibody titers.

参考文献 35

[1]	SHIRLEY M W, IVENS A, GRUBER A, et al. The Eimeria genome projects: a sequence of events[J]. Trends in Parasitology, 2004, 20(5): 199-201.
[2]	BLAKE D P, KNOX J, DEHAECK B, et al. Re-calculating the cost of coccidiosis in chickens[J]. Veterinary Research, 2020, 51(1): 115.
[3]	CHENGAT PRAKASHBABU B, THENMOZHI V, LIMON G, et al. Eimeria species occurrence varies between geographic regions and poultry production systems and may influence parasite genetic diversity[J]. Veterinary Parasitology, 2017, 233: 62-72.
[4]	DALLOUL R A, LILLEHOJ H S. Poultry coccidiosis: recent advancements in control measures and vaccine development[J]. Expert Review of Vaccines, 2006, 5(1): 143-163.
[5]	BLAKE D P, MARUGAN-HERNANDEZ V, TOMLEY F M. Spotlight on avian pathology: Eimeria and the disease coccidiosis[J]. Avian Pathology, 2021: 1-5.
[6]	GUO A J, CAI J P, GONG W, et al. Transcriptome analysis in chicken cecal epithelia upon infection by Eimeria tenella in vivo[J]. PLoS One, 2013, 8(5): e64236.
[7]	李超, 顾小龙, 卢春霞, 等. 我国鸡球虫病流行病学研究进展[J]. 寄生虫与医学昆虫学报, 2018, 25(4): 230-241.
	LI C, GU X L, LU C X, et al. Studies on the prevalence of chicken coccidiosis in China[J]. Acta Parasitologica et Medica Entomologica Sinica, 2018, 25(4): 230-241. (in Chinese with English abstract)
[8]	FOSSUM O, JANSSON D S, ETTERLIN P E, et al. Causes of mortality in laying hens in different housing systems in 2001 to 2004[J]. Acta Veterinaria Scandinavica, 2009, 51(1): 3.
[9]	CUI N, WANG X Z, WANG Q, et al. Effect of dual infection with Eimeria tenella and subgroup J avian leukosis virus on the cecal microbiome in specific-pathogen-free chicks[J]. Frontiers in Veterinary Science, 2017, 4: 177.
[10]	CLARKE L, FODEY T L, CROOKS S R H, et al. A review of coccidiostats and the analysis of their residues in meat and other food[J]. Meat Science, 2014, 97(3): 358-374.
[11]	NOACK S, CHAPMAN H D, SELZER P M. Anticoccidial drugs of the livestock industry[J]. Parasitology Research, 2019, 118(7): 2009-2026.
[12]	张思新, 汤新明, 李超, 等. 我国鸡球虫病疫苗研究进展[J]. 寄生虫与医学昆虫学报, 2018, 25(4): 254-261.
	ZHANG S X, TANG X M, LI C, et al. Progress in coccidiosis vaccine development for chickens in China[J]. Acta Parasitologica et Medica Entomologica Sinica, 2018, 25(4): 254-261. (in Chinese with English abstract)
[13]	GUPTA S K, SHUKLA P. Advanced technologies for improved expression of recombinant proteins in bacteria: perspectives and applications[J]. Critical Reviews in Biotechnology, 2016, 36(6): 1089-1098.
[14]	BLACKMAN M J, BANNISTER L H. Apical organelles of Api complexa: biology and isolation by subcellular fractionation[J]. Molecular and Biochemical Parasitology, 2001, 117(1): 11-25.
[15]	DASZAK P, BALL S J, PITTILO R M, et al. Ultrastructural evidence for dense granule exocytosis by first-generation merozoites of Eimeria tenella in vivo[J]. Parasitology Research, 1993, 79(3): 256-258.
[16]	汪明, 孔繁瑶. 毁灭泰泽球虫内生发育的超微结构研究 Ⅲ.小配子生殖与小配子[J]. 畜牧兽医学报, 1989, 20(1): 60-66.
	WANG M, KONG F Y. Ultrastructural studies on the endogenous development of Tyzzeria perniciosa, a coccidian parasite of pekin duck 3. microgametogenesis and the microgamete[J]. Chinese Journal of Animal and Veterinary Sciences, 1989, 20(1): 60-66. (in Chinese with English abstract)
[17]	MERCIER C, CESBRON-DELAUW M F. Toxoplasma secretory granules: one population or more?[J]. Trends in Parasitology, 2015, 31(2): 60-71.
[18]	CESBRON-DELAUW M F, GENDRIN C, TRAVIER L, et al. Apicomplexa in mammalian cells: trafficking to the parasitophorous vacuole[J]. Traffic, 2008, 9(5): 657-664.
[19]	PLATTNER F, SOLDATI-FAVRE D. Hijacking of host cellular functions by the Apicomplexa[J]. Annual Review of Microbiology, 2008, 62: 471-487.
[20]	SINAI A P. Biogenesis of and activities at the Toxoplasma gondii parasitophorous vacuole membrane[J]. Sub-Cellular Biochemistry, 2008, 47: 155-164.
[21]	MARTIN A M, LIU T, LYNN B C, et al. The Toxoplasma gondii parasitophorous vacuole membrane: transactions across the border[J]. The Journal of Eukaryotic Microbiology, 2007, 54(1): 25-28.
[22]	REZAEI F, SARVI S, SHARIF M, et al. A systematic review of Toxoplasma gondii antigens to find the best vaccine candidates for immunization[J]. Microbial Pathogenesis, 2019, 126: 172-184.
[23]	SCORZA T, D’SOUZA S, LALOUP M, et al. A GRA1 DNA vaccine primes cytolytic CD8⁺ T cells to control acute Toxoplasma gondii infection[J]. Infection and Immunity, 2003, 71(1): 309-316.
[24]	QUAN J H, CHU J Q, ISMAIL H A H A, et al. Induction of protective immune responses by a multiantigenic DNA vaccine encoding GRA7 and ROP1 of Toxoplasma gondii[J]. Clinical and Vaccine Immunology, 2012, 19(5): 666-674.
[25]	VAZINI H, GHAFARIFAR F, SHARIFI Z, et al. Evaluation of immune responses induced by GRA7 and ROP2 genes by DNA vaccine cocktails against acute toxoplasmosis in BALB/c mice[J]. Avicenna Journal of Medical Biotechnology, 2018, 10(1): 2-8.
[26]	SUN X F, WANG Z D, LI J P, et al. Evaluation of an indirect ELISA using recombinant granule antigen GRA1, GRA7 and soluble antigens for serodiagnosis of Toxoplasma gondii infection in chickens[J]. Research in Veterinary Science, 2015, 100: 161-164.
[27]	COSTA J G, VILARIÑO M J. Semiquantitative Dot Blot with the GRA8 antigen to differentiate the stages of toxoplasmosis infection[J]. Journal of Microbiological Methods, 2018, 149: 9-13.
[28]	TEIMOURI A, MODARRESSI M H, SHOJAEE S, et al. Development, optimization, and validation of an in-house Dot-ELISA rapid test based on SAG1 and GRA7 proteins for serological detection of Toxoplasma gondii infections[J]. Infection and Drug Resistance, 2019, 12: 2657-2669.
[29]	LIU X Y, WANG Z D, EL-ASHRAM S, et al. Toxoplasma gondii oocyst-driven infection in pigs, chickens and humans in northeastern China[J]. BMC Veterinary Research, 2019, 15(1): 366.
[30]	YBAÑEZ R H D, KYAN H, NISHIKAWA Y. Detection of antibodies against Toxoplasma gondii in cats using an immunochromatographic test based on GRA7 antigen[J]. The Journal of Veterinary Medical Science, 2020, 82(4): 441-445.
[31]	王英贺, 曹利利, 姚新华, 等. 弓形虫致密颗粒蛋白研究进展[J]. 动物医学进展, 2016, 37(8): 75-78.
	WANG Y H, CAO L L, YAO X H, et al. Progress on Toxoplasma gondii dense granule proteins[J]. Progress in Veterinary Medicine, 2016, 37(8): 75-78. (in Chinese with English abstract)
[32]	SIVALINGAM G N, SHEPHERD A J. An analysis of B-cell epitope discontinuity[J]. Molecular Immunology, 2012, 51(3/4): 304-309.
[33]	李仙春, 芦艳, 毛耀芳, 等. 鸡Prnp基因原核表达载体的构建及其在大肠埃希菌中的表达[J]. 浙江农业学报, 2020, 32(12): 2138-2146.
	LI X C, LU Y, MAO Y F, et al. Construction of prokaryotic expression vector of chicken Prnp gene and expression in Escherichia coli[J]. Acta Agriculturae Zhejiangensis, 2020, 32(12): 2138-2146. (in Chinese with English abstract)
[34]	HU D D, TANG X M, BEN MAMOUN C, et al. Efficient single-gene and gene family editing in the api complexan parasite Eimeria tenella using CRISPR-Cas9[J]. Frontiers in Bioengineering and Biotechnology, 2020, 8: 128.
[35]	MU B J, MENG Y J, LIU X X, et al. Research note: transcriptomic analysis of LMH cells in response to the overexpression of a hypothetical protein identified in Eimeria tenella SD-01 strain[J]. Poultry Science, 2022, 101(11): 102109.

鸡柔嫩艾美耳球虫2种假定致密颗粒蛋白基因的克隆与表达

Cloning and expression analysis of two hypothetical dense granule protein genes of Eimeria tenella in chickens

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 9

参考文献 35

相关文章 15

编辑推荐

Metrics

本文评价

[1]	宋传生, 康晓飞, 樊庆忠, 王俊刚, 石雪, 张子汝, 谭青青, 曾小娇, 刘芳, 李英赛, 侯常跃. 枣疯病植原体胸苷激酶基因的克隆、序列分析与原核表达[J]. 浙江农业学报, 2023, 35(8): 1763-1772.
[2]	刘琪, 曹影丽, 魏宁波, 杨侃侃, 梁月巧, 宋祥军, 邵颖, 涂健, 祁克宗. 鸡DDX41基因克隆表达、细胞定位及其在禽腺病毒4型感染调控天然免疫中的作用[J]. 浙江农业学报, 2023, 35(5): 1028-1036.
[3]	薛姣雄, 赵婷芳, 张倩, 唐青海, 高翠翠, 赵铖, 张妍, 全飞杨, 刘婷, 杨灿, 杨海, 王文秀. 犬冠状病毒S1蛋白特异性小分子抗体Fab的制备[J]. 浙江农业学报, 2023, 35(11): 2568-2583.
[4]	唐国亮, 张玉宝, 王若愚, 王亚军, 赵霞, 苏学思, 金卫杰. 魔芋花叶病毒衣壳蛋白的原核表达和多克隆抗体制备[J]. 浙江农业学报, 2022, 34(11): 2471-2481.
[5]	朱寅初, 王宏宇, 云涛, 华炯钢, 叶伟成, 倪征, 陈柳, 张存. 浙江地区鹅星状病毒分离鉴定及其衣壳蛋白多克隆抗体的制备[J]. 浙江农业学报, 2022, 34(10): 2149-2159.
[6]	赵秀平, 王双, 闫星伊, 段强, 张帅, 陈永胜, 李国瑞. 稻瘟病菌MGG-01005的表达纯化与生物信息学分析[J]. 浙江农业学报, 2021, 33(3): 470-478.
[7]	苏学思, 张玉宝, 王若愚, 王亚军, 唐国亮, 金卫杰. 车前草花叶病毒衣壳蛋白的原核表达和多克隆抗体制备[J]. 浙江农业学报, 2021, 33(1): 104-111.
[8]	刘金玉, 黄鹰. 粟酒裂殖酵母SpTrz2蛋白全长和N端的原核表达与多克隆抗体制备[J]. 浙江农业学报, 2021, 33(1): 34-42.
[9]	黄小珍, 乔中全, 曾慧杰, 李永欣, 何钢, 王晓明. 紫薇花器官发育调控基因LiFUL1的分离与表达分析[J]. 浙江农业学报, 2020, 32(7): 1206-1214.
[10]	陈韫陆, 单颖, 罗浩, 徐计东, 赵灵燕, 方维焕, 李肖梁. 猪Ⅲ型干扰素原核表达及其抗病毒效果研究[J]. 浙江农业学报, 2020, 32(5): 779-788.
[11]	蒲路莎, 苏世博, 陈肖韩, 赵丽丽, 陈洪岩. 鹅源星状病毒ORF2基因原核表达及遗传进化分析[J]. 浙江农业学报, 2020, 32(5): 789-797.
[12]	王元红, 邢雪, 李传峰, 朱杰, 王勇, 刘光清. 猫传染性腹膜炎病毒AH1905株N基因的生物信息学分析及原核表达[J]. 浙江农业学报, 2020, 32(3): 406-414.
[13]	王小朋, 赵靓, 刘自敏, 白彩霞, 杨侃侃, 张达, 孙裴, 蒋书东, 李永东, 王勇. 猪细小病毒7型Cap基因原核表达与生物信息学分析[J]. 浙江农业学报, 2020, 32(2): 200-209.
[14]	李仙春, 芦艳, 毛耀芳, 杨海峰, 余海山, 马永华, 万学瑞. 鸡Prnp基因原核表达载体的构建及其在大肠埃希菌中的表达[J]. 浙江农业学报, 2020, 32(12): 2138-2146.
[15]	陈章, 吴华健, 毛天骄, 韩业芹, 孙裴, 魏建忠, 李东风, 李郁. 猪链球菌2型制苗用菌株的筛选[J]. 浙江农业学报, 2020, 32(1): 57-64.