鸡传染性法氏囊病病毒BJQ902株感染性克隆的构建

doi:10.16431/j.cnki.1671-7236.2026.02.036

摘要/Abstract

摘要：

目的利用反向遗传操作技术构建鸡传染性法氏囊病病毒（Infectious bursal disease virus，IBDV）BJQ902株感染性克隆，为疫苗的开发及病毒复制和致病机制研究提供重要工具。方法以BJQ902株全基因组序列为基础，利用PCR技术扩增IBDV BJQ902株基因组A、B片段，并在A、B片段两端分别引入自裂解核酶RiboJ和HDVrz序列，利用同源重组技术将带有核酶结构的A、B片段插入到载体pCAGGS上，构建重组质粒pCAGGS-BJQ-RAH和pCAGGS-BJQ-RBH，将重组质粒pCAGGS-BJQ-RAH和pCAGGS-BJQ-RBH共转染DF-1细胞，连续盲传3代拯救重组BJQ902株（rBJQ902）。将BJQ902株及rBJQ902株感染DF-1细胞，利用PCR、Western blotting和间接免疫荧光试验（IFA）鉴定rBJQ902株。结果成功获得了BJQ902株基因组A、B片段，大小分别为3 260和2 827 bp；将自裂解核酶RiboJ和HDVrz序列分别连在A、B片段两端，大小分别为3 436和3 033 bp；将带有核酶序列的A、B片段分别连接到pCAGGS载体上，成功获得了重组质粒pCAGGS-BJQ-RAH和pCAGGS-BJQ-RBH；在DF-1细胞中连续盲传3代获得rBJQ902株；与亲本毒株BJQ902相比，感染DF-1细胞48 h后，rBJQ902株也可扩增得到VP2片段；rBJQ902株的病变与亲本株一致；Western blotting和IFA都可检测到rBJQ902株VP2蛋白的表达，该毒株在细胞培养中展现出与亲本病毒相似的生物学特性。结论本研究成功获得了BJQ902株的感染性克隆毒株rBJQ902，为深入研究 IBDV 的生物学特性及致病机制提供了技术支持，为后续病毒学研究奠定基础。

关键词: 传染性法氏囊病病毒（IBDV）; 感染性克隆; 病毒拯救; 核酶

Abstract:

Objective The purpose of this study was to construct infectious clone of Infectious bursal disease virus （IBDV） BJQ902 strain using reverse genetics technology， and provide important tools for vaccine development and the research of viral replication and pathogenic mechanisms. Method Based on the whole genome sequence of BJQ902 strain， PCR was used to amplify A and B fragments of IBDV BJQ902 strain’s genome. The RiboJ and HDVrz sequences were introduced at both ends of A and B fragments， respectively. Homologous recombination technology was employed to insert A and B fragments with ribozyme structures into vector pCAGGS， constructing the recombinant plasmids pCAGGS-BJQ-RAH and pCAGGS-BJQ-RBH. These vectors were co-transfected into DF-1 cells， and rBJQ902 strain was rescued after three consecutive passages in blind transmission. DF-1 cells were infected with both BJQ902 and rBJQ902 strains， and rBJQ902 strain was identified using PCR， Western blotting and indirect immunofluorescence assay （IFA）. Result The BJQ902 strain genome A and B fragments were successfully obtained， with sizes of 3 260 and 2 827 bp， respectively. The self-splicing ribozymes RiboJ and HDVrz sequences were ligated to the ends of fragment A and B， with sizes of 3 436 and 3 033 bp， respectively. The ribozyme-containing fragments A and B were then ligated to the pCAGGS vector， resulting in the successful acquisition of the recombinant plasmids pCAGGS-BJQ-RAH and pCAGGS-BJQ-RBH. rBJQ902 strain was obtained by blind passage for three consecutive generations in DF-1 cells. Compared with the parent strain BJQ902， rBJQ902 strain could also amplify the VP2 fragment after infecting DF-1 cells for 48 h. The lesions caused by rBJQ902 strain were consistent with those of the parent strain. Both Western blotting and IFA could detect the expression of VP2 protein in rBJQ902 strain， demonstrating that this strain exhibited similar biological characteristics to the parent virus during cell culture. Conclusion This study successfully obtained the infectious clone strain rBJQ902 of BJQ902 strain， providing technical support for in-depth research on the biological characteristics and pathogenic mechanism of IBDV and laying a foundation for subsequent virological research.

Key words: Infectious bursal disease virus （IBDV）; infectious clone; virus rescue; ribozyme

中图分类号:

S852.65⁺9.4

吕然, 杨鸿玮, 涂敏, 孟昭英, 靳换, 章振华. 鸡传染性法氏囊病病毒BJQ902株感染性克隆的构建[J]. 中国畜牧兽医, 2026, 53(2): 916-925.

LYU Ran, YANG Hongwei, TU Min, MENG Zhaoying, JIN Huan, ZHANG Zhenhua. Construction of an Infectious Clone of Chicken Infectious Bursal Disease Virus Strain BJQ902[J]. China Animal Husbandry & Veterinary Medicine, 2026, 53(2): 916-925.

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引物名称 Primer name	片段Segment	引物序列 Primer sequences （5'→3'）	退火温度 Annealing temperature/℃	用途 Purpose
IBDV-A-F	A	GGATACGATCGGTCTGACCC	55	全基因组扩增
IBDV-A-R	A	GGGGACCCGCGAACGGATCCA	55	全基因组扩增
pCAG-R-SeqA-F	A	GGCAAAGAATTCCTCGAGAGCTGTCACCGGATGTGCT	58	引入RiboJ核酶序列
pCAG-R-SeqA-R	A	GTTGGGTTTGATCTTGCAGGTTTGT	58	引入RiboJ核酶序列
SeqA-H-pCAG-F	A	AGGGCTGTCTCTGATGAGGACCTTG	58	引入HDVrz核酶序列
SeqA-H-pCAG-R	A	AGGAGTGAATTCCTCGAGTCGAGGCGGATCCGAGCTC	58	引入HDVrz核酶序列
IBDV-B-F	B	GGATACGATGGGTCTGACCCT	55	全基因组扩增
IBDV-B-R	B	GGGGGCCCCCGCAGGCGAAGG	55	全基因组扩增
pCAG-R-SeqB-F	B	GGCAAAGAATTCCTCGAGAGCTGTCACCGGATGTGCT	58	引入RiboJ核酶序列
pCAG-R-SeqB-R	B	CAGAGGGTCAGACCCATCGT	58	引入RiboJ核酶序列
SeqB-H-pCAG-F	B	CCCGGCCTTCGCCTGCGGGG	58	引入HDVrz核酶序列
SeqB-H-pCAG-R	B	AGGAGTGAATTCCTCGAGTCGAGGCGGATCCGAGCTC	58	引入HDVrz核酶序列
VP2-F	A	ATGACAAACCTGCAAGATCAA	55	鉴定VP2
VP2-R	A	CCTTATGGCCCGGATTATGTC	55	鉴定VP2