表达非洲猪瘟病毒CD2v与P12蛋白的重组伪狂犬病毒的构建

doi:10.13560/j.cnki.biotech.bull.1985.2021-0024

摘要/Abstract

摘要：

非洲猪瘟（African swine fever,ASF）与伪狂犬病（pseudorabies,PR）分别由非洲猪瘟病毒（African swine fever virus,ASFV）与伪狂犬病毒感染（Pseudorabies virus,PRV）引起的猪（或部分猪）高致死、传染性疾病。目前非洲猪瘟无商业化疫苗,两种疾病均给养猪业造成了巨大的经济损失。本研究以PRV-Fa经典毒株为载体通过CRISPR/Cas9（clustered regularly interspaced short palindromic repeats/Cas9）基因编辑技术将ASFV（Pig/HLJ/18）CD2v（EP402R）与p12（O61R）基因分别插入PRV-Fa株TK（UL23）及gI（US7）基因位点中,成功构建TK及gI缺失且重组表达ASFV CD2v与P12的重组弱毒株PRV-∆gI-（P12）-∆TK-（CD2v）。通过基因测序、蛋白质免疫印迹（Western blot）与免疫荧光表明重组毒株遗传性状稳定且CD2v与P12在Vero细胞中稳定表达。通过一步生长曲线、噬斑生长测定、小鼠毒力测试及小鼠病理学评估表明与野生型毒株PRV-Fa相比重组毒株毒力明显减弱,对小鼠不具致死能力,该重组毒株为研究预防PR及ASF的新型疫苗提供了新的选择。

关键词: 非洲猪瘟, 伪狂犬病毒, CD2v, P12, 疫苗, 重组疫苗

Abstract:

ASF（African swine fever）and PR（pseudorabies）both are highly lethal and infectious diseases for pigs infected by African swine fever virus（ASFV）and pseudorabies virus（PRV）,respectively. Both diseases have caused huge economic losses to the pig breeding industry and no commercial vaccine is available for preventing African swine fever. In this study,we inserted the ASFV（Pig/HLJ/18）CD2v（EP402R）and p12（O61R）genes into the PRV-Fa TK（UL23）and gI（US7）gene loci,respectively by CRISPR/Cas9 technology and classical viral strain PRV-Fa,and successfully constructed a recombinant attenuated strain PRV-∆gI-（P12）-∆TK-（CD2v）with deletion of TK and gI as well as expression of CD2v and P12. Gene sequencing,Western blotting and immunofluorescence analyses showed that the genetic traits of the recombinant strain were stable,and the CD2v and P12 were stably expressed in Vero cells. The one-step growth curve,plaque growth assay,mouse virulence test and pathological evaluation indicated that the virulence of the recombinant strain was significantly attenuated and not lethal to mice compared with the wild-type strain PRV-Fa. Therefore,the recombinant strain will become a new selection for researching of vaccine to prevent PR and ASF.

Key words: African swine fever, pseudorabies, CD2v, P12, vaccine, recombination vaccine

梁旺旺, 李成龙, 陈文智, 丰志华, 蔡少丽, 陈骐. 表达非洲猪瘟病毒CD2v与P12蛋白的重组伪狂犬病毒的构建[J]. 生物技术通报, 2021, 37(12): 132-140.

LIANG Wang-wang, LI Cheng-long, CHEN Wen-zhi, FENG Zhi-hua, CAI Shao-li, CHEN Qi. Construction of Recombinant Pseudorabies Virus Expressing CD2v and P12 Proteins of African Swine Fever Virus[J]. Biotechnology Bulletin, 2021, 37(12): 132-140.

图/表 7

表1 靶向CD2v与p12基因的sgRNA序列

Table 1 sgRNA sequences targetting CD2v and p12 gene

Primer	Primer sequence
sgRNA-TK	CGCGGGGTCGTACGTGCTGC
sgRNA-gI	TGCCCGAGCCGATGGCGTAC

Fig.1 重组毒株PRV-△gI-（p12）-△TK-（CD2v）构建示意图

Fig.1 Schematic diagram of constructing recombinant strain（PRV-△gI-（p12）-△TK-（CD2v））

表2 PCR扩增引物与验证引物

Table 2 PCR primers for amplification and verfication

Primer	Primer sequence
3.1-CD2v	GTCGACGCCACGAACAC
3.1-CD2v	TCTAGAACGTGTTGACCAGCAT
3.1-p12	GTCGACATGATGATGGTGGC
3.1-p12	TCTAGAACGACGATCGTGGG
PRV-TK	CTCGATGACGAAGCACAGGT
PRV-TK	TTGACCAGCATGGCGTAGAC
PRV-p12	GACGCTGCTGTTTCTGGAGG
PRV-p12	TCTAGAACGTGTTGACCAGCAT
PRV-CD2v-F	CGCCCAAGAAGATCAGGAGG
PRV-CD2v-F	TAGGGGGCGTACTTGGCATA
PRV-CD2v-R	CACAGTCCCCGAGAAGTTGG
PRV-CD2v-R	CCCAGCACCATGGCTATCTT
PRV-p12-F	CTCGATGACGAAGCACAGGT
PRV-p12-F	TAGGGGGCGTACTTGGCATA
PRV-p12-R	AGCATGATGACACCACTTCCA
PRV-p12-R	TGATGTCCCCGACGATGAAG

图2 同源重组片段制备及重组位点验证 A:M:DL5000 DNA maker,1:以pcDNA3.1-PRV-TK-P12为模板通过PCR扩增出带同源臂的p12片段,2:以pcDNA3.1-PRV-gI-CD2v为模板通过PCR扩增出带同源臂的CD2v片段,3和4:空白对照（以水为模板）。B:M:DL5000 DNA maker,1:以野生型PRV基因组为模板通过PCR扩增的TK基因片段,2:以重组病毒PRV-∆gI-（p12）-∆TK-（CD2v）基因组为模板通过PCR扩增的CD2v插入后的TK基因片段,3:以野生型PRV基因组为模板通过PCR扩增的gI基因片段,泳道4为以重组病毒PRV-∆gI-（p12）-∆TK-（CD2v）基因组为模板通过PCR扩增的CD2v插入后的gI基因片段。C:M:DL2000 DNA maker,1:以重组病毒PRV-∆gI-（p12）-∆TK-（CD2v）基因组为模板通过PCR扩增含部分上游同源臂及CD2v基因片段,2:以重组病毒PRV-∆gI-（p12）-∆TK-（CD2v）基因组为模板通过PCR扩增含部分下游同源臂及CD2v基因片段,3:以重组病毒PRV-∆gI-（p12）-∆TK-（CD2v）基因组为模板通过PCR扩增含部分上游同源臂及gI基因片段,4:以重组病毒PRV-∆gI-（p12）-∆TK-（CD2v）基因组为模板通过PCR扩增含部分下游同源臂及gI基因片段。D:图2-C中PCR产物测序结果

Fig.2 Preparation of homologous recombinant fragment and verification of recombination sites A:M:DL 5000 DNA marker. 1:PCR results of p12 gene with both sides donor from pcDNA3.1-PRV-TK-P12. 2:PCR results of CD2v gene with both sides donor from pcDNA3.1-PRV-gI-CD2v. 3 and 4:Blank control（water as template）. B:M:DL 5000 DNA marker. 1:PCR results of TK gene from wild-type PRV. 2:PCR results of TK gene from PRV-∆gI-（p12）-∆TK-（CD2v）. 3:PCR results of gI gene from wild-type PRV. 4:PCR results of gI gene from PRV-∆gI-（p12）-∆TK-（CD2v）. C:M:DL2000 marker. 1:PCR results of CD2v gene with 5' donor from PRV-∆gI-（p12）-∆TK-（CD2v）. 2:PCR results of CD2v gene with 3' donor from PRV-∆gI-（p12）-∆TK-（CD2v）. 3:PCR results of gI gene with 5' Donor from PRV-∆gI-（p12）-∆TK-（CD2v）. 4:PCR results of gI gene with 3' donor from PRV-∆gI-（p12）-∆TK-（CD2v）. D:Sequencing analysis from Fig.2-C

图3 遗传稳定性鉴定 A:通过PCR技术检测重组病毒稳定遗传情况。M:DL5000 DNA maker;1、3、5:分别为重组病毒PRV-∆gI-（p12）-∆TK-（CD2v）在10、20、30代p12基因PCR结果;2、4、6:分别为重组病毒PRV-∆gI-（p12）-∆TK-（CD2v）在10、20、30代CD2v基因PCR结果。B:通过Western blot技术检测感染PRV-∆gI-（p12）-∆TK-（CD2v）的Vero细胞中CD2v蛋白表达情况。M2:蛋白 Marker;C1:Vero细胞对照组;C2:感染PRV-Fa组;C3:感染PRV-∆gI-（p12）-∆TK-（CD2v）组。C:通过免疫荧光技术检测EGFP与mCherry基因在PRV-∆gI-（p12）-∆TK-（CD2v）表达情况

Fig.3 Identification of genetic stability A:PCR detection for genetic stability of recombinant virus. M:DL5000 maker; 1,3 and 5:the PCR result of p12 gene from 10th,20th and 30th Vero cells infected with PRV-∆gI-（p12）-∆TK-（CD2v）recombinant virus,respectively; 2,4 and 6:the PCR result of CD2v gene from 10th,20th and 30th Vero cells infected with PRV-∆gI-（p12）-∆TK-（CD2v）,respectively. B:The expression of CD2v protein in Vero cells infected with PRV-∆gI-（p12）-∆TK-（CD2v）recombinant virus is confirmed by Western blot. Lane M2:Protein marker; C1:control（Vero cells）; C2:Vero cells infected with PRV-Fa; C3:Vero cells infected with PRV-∆gI-（p12）-∆TK-（CD2v）. C:Expression of EGFP and mCherry in PRV-∆gI-（p12）-∆TK-（CD2v）via immunofluorescence analysis

图4 重组病毒一步生长曲线与小鼠生存曲线 A:未感染病毒的Vero细胞的结晶紫染色;B:感染PRV-Fa病毒的Vero细胞结晶的紫染色;C:感染PRV-∆gI-（p12）-∆TK-（CD2v）病毒的Vero细胞结晶的紫染色;D:噬班面积统计结果;E:病毒在Vero 细胞中扩增后按照12 h、24 h、36 h、48 h、72 h的时间梯度收样后用 Karber法计算绘制一次性生长曲线;F:5周龄ICR小鼠右后腿肌肉注射病毒逐天观察小鼠存活情况绘制生存曲线（n=15）。各组数据差异性分析用 Unpaired t-test（Graphpad Prism 5.0,Graphpad software,San Diego,CA,USA）,*P<0.05;**P<0.01;***P<0.001;ns（not significant）

Fig.4 One-step growth curve of recombinant viruses and mice’s survival curve A:Crystal violet staining of Vero cells（mock）. B:Crystal violet staining of Vero cells infected with PRV-Fa virus. C:Crystal violet staining of Vero cells infected with PRV-∆gI-（p12）-∆TK-（CD2v）virus. D:Statistical results of the plaque area. E:Virus sample after the amplification in Vero cells was collected at 12,24,36,and 48 h,and the virus titer was calculated by the Karber method to draw a one-step growth curve. F:Five-week-old ICR mice were injected with viruses into the right hind leg to observe mice’s survival daily（n = 15/each group）. All data were analysed by unpaired t-test（GraphPad Prism 5.0,GraphPad Software,San Diego,CA,USA）,*P<0.05;**P<0.01;***P<0.001;ns（not significant）

图5 小鼠组织病理学切片 A:心脏HE染色;B:肝脏HE染色;C:脾脏HE染色,PRV-∆gI-（p12）-∆TK-（CD2v）感染组的脾脏有大量的免疫细胞聚集;D:肺脏HE染色,PRV-Fa感染组的肺脏有明显肿大;E:肾脏HE染色;F:大脑HE染色,PRV-Fa感染组的大脑内有免疫细胞浸润

Fig.5 Histopathological sections of mice tissues A:HE（Hematoxylin-Eosin）staining of heart. B:HE staining of liver. C:HE staining of spleen,there are a lot of immune cells accumulated in the spleens of PRV-∆gI-（p12）-∆TK-（CD2v）infected group. D:HE（Hematoxylin-Eosin）staining of lung,the lung of PRV-Fa infected group was swollen obviously. E:HE staining of kidney. F:HE staining of brain,the brain of PRV-Fa infected group showed a large number of immune cell accumulated

参考文献 25

[1]	Galindo I, Alonso C. African swine fever virus:a review[J]. Viruses, 2017, 9(5). DOI: 10. 3390/v9050103. doi: 10. 3390/v9050103
[2]	Quembo CJ, Jori F, Vosloo W, et al. Genetic characterization of African swine fever virus isolates from soft ticks at the wildlife/domestic interface in Mozambique and identification of a novel genotype[J]. Transbound Emerg Dis, 2018, 65(2):420-431. doi: 10.1111/tbed.12700 pmid: 28921895
[3]	Zhou X, Li N, et al. Emergence of African swine fever in China, 2018[J]. Transbound Emerg Dis, 2018, 65(6):1482-1484. doi: 10.1111/tbed.2018.65.issue-6 URL
[4]	Sánchez EG, Pérez-Núñez D, Revilla Y. Mechanisms of entry and endosomal pathway of African swine fever virus[J]. Vaccines(Basel), 2017, 5(4):E42.
[5]	Alejo A, Matamoros T, Guerra M, et al. A proteomic atlas of the African swine fever virus particle[J]. J Virol, 2018, 92(23):e01293-e01218.
[6]	Goatley LC, Dixon LK. Processing and localization of the African swine fever virus CD2v transmembrane protein[J]. J Virol, 2011, 85(7):3294-3305. doi: 10.1128/JVI.01994-10 URL
[7]	Monteagudo PL, Lacasta A, López E, et al. BA71ΔCD2:a new recombinant live attenuated African swine fever virus with cross-protective capabilities[J]. J Virol, 2017, 91:e01058-17.
[8]	Angulo A, Viñuela E, Alcamí A. Inhibition of African swine fever virus binding and infectivity by purified recombinant virus attachment protein p12[J]. J Virol, 1993, 67(9):5463-5471. pmid: 8350406
[9]	Jancovich JK, Chapman D, Hansen DT, et al. Immunization of pigs by DNA prime and recombinant vaccinia virus boost to identify and rank African swine fever virus immunogenic and protective proteins[J]. Journal of Virology, 2018, 92(8):e02219-17.
[10]	Pérez-Núñez D, Sunwoo SY, et al. Evaluation of a viral DNA-protein immunization strategy against African swine fever in domestic pigs[J]. Vet Immunol Immunopathol, 2019, 208:34-43. doi: 10.1016/j.vetimm.2018.11.018 URL
[11]	Lopera-Madrid J, Osorio JE, He Y, et al. Safety and immunogenicity of mammalian cell derived and Modified Vaccinia Ankara vectored African swine fever subunit antigens in swine[J]. Vet Immunol Immunopathol, 2017, 185:20-33. doi: 10.1016/j.vetimm.2017.01.004 URL
[12]	Pomeranz LE, Reynolds AE, Hengartner CJ. Molecular biology of pseudorabies virus:impact on neurovirology and veterinary medicine[J]. Microbiol Mol Biol Rev, 2005, 69(3):462-500. doi: 10.1128/MMBR.69.3.462-500.2005 URL
[13]	Oláh P, Tombácz D, Póka N, et al. Characterization of pseudorabies virus transcriptome by Illumina sequencing[J]. BMC Microbiol, 2015, 15:130. doi: 10.1186/s12866-015-0470-0 URL
[14]	Tong W, Li GX, Liang C, et al. A live, attenuated pseudorabies virus strain JS-2012 deleted for gE/gI protects against both classical and emerging strains[J]. Antivir Res, 2016, 130:110-117. doi: 10.1016/j.antiviral.2016.03.002 pmid: 26946112
[15]	Tang YD, Liu JT, Wang TY, et al. Live attenuated pseudorabies virus developed using the CRISPR/Cas9 system[J]. Virus Res, 2016, 225:33-39. doi: 10.1016/j.virusres.2016.09.004 URL
[16]	Wu KK, Liu JM, Wang LX, et al. Current state of global African swine fever vaccine development under the prevalence and transmission of ASF in China[J]. Vaccines, 2020, 8(3):531. doi: 10.3390/vaccines8030531 URL
[17]	Ramirez-Medina E, Vuono E, O’Donnell V, et al. Differential effect of the deletion of African swine fever virus virulence-associated genes in the induction of attenuation of the highly virulent Georgia strain[J]. Viruses, 2019, 11(7):599. doi: 10.3390/v11070599 URL
[18]	Revilla Y, Pérez-Núñez D, Richt JA. African swine fever virus biology and vaccine approaches[J]. Adv Virus Res, 2018, 100:41-74.
[19]	Gaudreault NN, Richt JA. Subunit vaccine approaches for African swine fever virus[J]. Vaccines, 2019, 7(2):56. doi: 10.3390/vaccines7020056 URL
[20]	Malogolovkin A, Burmakina G, Tulman ER, et al. African swine fever virus CD2v and C-type lectin gene loci mediate serological specificity[J]. J Gen Virol, 2015, 96(4):866-873. doi: 10.1099/jgv.0.000024 URL
[21]	Ruiz-Gonzalvo F, Rodríguez F, Escribano JM. Functional and immunological properties of the baculovirus-expressed hemagglutinin of African swine fever virus[J]. Virology, 1996, 218(1):285-289. pmid: 8615037
[22]	Jiang YB, Fang LR, Xiao SB, et al. Construction and immunogenicity of recombinant pseudorabies virus expressing the modified GP5m protein of porcine reproduction and respiratory syndrome virus[J]. Front Biol China, 2007, 2(1):85-91. doi: 10.1007/s11515-007-0015-5 URL
[23]	Feng ZH, Chen JH, Liang WW, et al. The recombinant pseudorabies virus expressing African swine fever virus CD2v protein is safe and effective in mice[J]. Virol J, 2020, 17(1):180. doi: 10.1186/s12985-020-01450-7 URL
[24]	Qiu HJ, Tian ZJ, Tong GZ, et al. Protective immunity induced by a recombinant pseudorabies virus expressing the GP5 of porcine reproductive and respiratory syndrome virus in piglets[J]. Vet Immunol Immunopathol, 2005, 106(3/4):309-319. doi: 10.1016/j.vetimm.2005.03.008 URL
[25]	Abid M, Teklue T, Li YF, et al. Generation and immunogenicity of a recombinant pseudorabies virus co-expressing classical swine fever virus E2 protein and porcine Circovirus type 2 capsid protein based on fosmid library platform[J]. Pathogens, 2019, 8(4):E279.