Efficient Cross-Screening and Characterization of Monoclonal Antibodies against Marek's Disease Specific Meq Oncoprotein Using CRISPR/Cas9-Gene-Edited Viruses.
Efficient Cross-Screening and Characterization of Monoclonal Antibodies against Marek's Disease Specific Meq Oncoprotein Using CRISPR/Cas9-Gene-Edited Viruses.
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DOI：
10.3390/v15040817
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发表时间：
2023-03-23
期刊：
影响因子：
--
通讯作者：
Luo J
中科院分区：
文献类型：
--
作者：
Teng M;Liu JL;Luo Q;Zheng LP;Yao Y;Nair V;Zhang GP;Luo J
由致病性马立克氏病病毒1型(−-1)引起的马立克氏病(MD)是家禽最重要的肿瘤性疾病之一。MDVMEQ1编码的独特−蛋白是主要的癌蛋白，MEQ1特异性单抗的存在对揭示MDVMEQ的致病机制和致癌机制具有重要意义。以MEQ蛋白亲水性保守区域合成的多肽为免疫原，结合杂交瘤技术和交叉免疫荧光试验对CRISPR/−-基因编辑产生的MEQ缺失的MDVCas1病毒进行初步筛选，共产生5株阳性杂交瘤细胞。其中2A9、5A7、7F9和8G11四株杂交瘤细胞分泌抗MEQ的特异性抗体，高表达MEQ的293T细胞的IFA染色进一步证实了这一点。用这些抗体染色的细胞的共聚焦显微镜分析证实了MEQ在MDV感染的CEF细胞和MDV转化的MSB-1细胞中的核定位。2A9-B12和8G11-B2两株杂交瘤细胞株对不同毒力的−-1株的MEQ蛋白具有较高的特异性。我们利用人工合成的多肽免疫，结合CRISPR/Cas9基因编辑病毒的交叉IFA染色，为下一代针对病毒蛋白的特异性单抗提供了一种新的有效方法。
Marek’s disease (MD) caused by pathogenic Marek’s disease virus type 1 (MDV−1) is one of the most important neoplastic diseases of poultry. MDV−1-encoded unique Meq protein is the major oncoprotein and the availability of Meq-specific monoclonal antibodies (mAbs) is crucial for revealing MDV pathogenesis/oncogenesis. Using synthesized polypeptides from conserved hydrophilic regions of the Meq protein as immunogens, together with hybridoma technology and primary screening by cross immunofluorescence assay (IFA) on Meq-deleted MDV−1 viruses generated by CRISPR/Cas9-gene editing, a total of five positive hybridomas were generated. Four of these hybridomas, namely 2A9, 5A7, 7F9 and 8G11, were further confirmed to secrete specific antibodies against Meq as confirmed by the IFA staining of 293T cells overexpressing Meq. Confocal microscopic analysis of cells stained with these antibodies confirmed the nuclear localization of Meq in MDV-infected CEF cells and MDV-transformed MSB-1 cells. Furthermore, two mAb hybridoma clones, 2A9-B12 and 8G11-B2 derived from 2A9 and 8G11, respectively, displayed high specificity for Meq proteins of MDV−1 strains with diverse virulence. Our data presented here, using synthesized polypeptide immunization combined with cross IFA staining on CRISPR/Cas9 gene-edited viruses, has provided a new efficient approach for future generation of specific mAbs against viral proteins.
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影响因子：
1.4
作者：
Kennedy DA;Cairns C;Jones MJ;Bell AS;Salathé RM;Baigent SJ;Nair VK;Dunn PA;Read AF
通讯作者：
Read AF
影响因子：
4.3
作者：
Deng, Qiaomu;Shi, Mengya;Wei, Ping
通讯作者：
Wei, Ping
DOI：
10.1099/jgv.0.001673
发表时间：
2021-10
期刊：
The Journal of general virology
影响因子：
--
作者：
Gatherer D;Depledge DP;Hartley CA;Szpara ML;Vaz PK;Benkő M;Brandt CR;Bryant NA;Dastjerdi A;Doszpoly A;Gompels UA;Inoue N;Jarosinski KW;Kaul R;Lacoste V;Norberg P;Origgi FC;Orton RJ;Pellett PE;Schmid DS;Spatz SJ;Stewart JP;Trimpert J;Waltzek TB;Davison AJ
通讯作者：
Davison AJ
DOI：
10.1073/pnas.0404508101
发表时间：
2004-08-10
影响因子：
11.1
作者：
Lupiani, B;Lee, LF;Reddy, SM
通讯作者：
Reddy, SM
影响因子：
3.3
作者：
Shamblin, CE;Greene, N;Parcells, MS
通讯作者：
Parcells, MS