Chowdhury S I, Ross C S, Lee B J, Hall V, Chu H J
Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan 66506, USA.
Am J Vet Res. 1999 Feb;60(2):227-32.
To construct and characterize a recombinant glycoprotein (g)E gene-deleted bovine herpesvirus (BHV) type 1 (BHV-1).
The BHV-1 gEgene-coding region and the flanking upstream and downstream sequences were cloned. The aforementioned cloned DNA was digested with suitable enzymes to release the amino terminal two thirds of that region, and was ligated to the beta-galactosidase (beta-gal) gene. The resulting plasmid DNA was cotransfected with DNA from full-length, wild-type (WT), BHV-1 Cooper strain of the virus. Recombinant viruses expressing beta-gal (blue plaques) were plaque purified and assayed further by blot hybridization for genetic characterization and by immunoblotting for reactivity against BHV-1 gE peptide-specific rabbit polyclonal antibody. One recombinant virus, gEdelta3.1IBR, was characterized in vitro and in vivo. The ability of the recombinant virus to induce BHV-1 neutralizing antibodies in infected calves was investigated by plaque-reduction tests.
The gEdelta3.1IBR virus contained a deletion in the viral gE gene-coding sequences where a stable chimeric reporter (beta-gal) gene was inserted. One-step growth kinetics and virus yield of the recombinant and parent viruses were similar, but early after infection, the recombinant virus yield was comparatively less. After intranasal inoculation, the recombinant gEdelta3.1IBR virus replicated in the upper respiratory tract of calves, but the amount of progeny viruses produced was hundredsfold reduced, and duration of virus shedding was shorter. Results of in vivo calf experiments and serum neutralization tests indicated that deleting the gE gene has little effect on inducing neutralizing antibodies against BHV-1, but is sufficient to reduce BHV-1 virulence in calves.
构建并鉴定一种缺失重组糖蛋白（g）E基因的1型牛疱疹病毒（BHV - 1）。
克隆BHV - 1 gE基因编码区及其上下游侧翼序列。用合适的酶消化上述克隆的DNA，释放该区域氨基末端的三分之二，并与β - 半乳糖苷酶（β - gal）基因连接。将所得质粒DNA与全长野生型（WT）BHV - 1库珀株病毒的DNA共转染。对表达β - gal的重组病毒（蓝色噬斑）进行噬斑纯化，并通过印迹杂交进一步进行基因鉴定，通过免疫印迹检测其对BHV - 1 gE肽特异性兔多克隆抗体的反应性。对一种重组病毒gEdelta3.1IBR进行了体内外鉴定。通过噬斑减少试验研究重组病毒在感染犊牛中诱导BHV - 1中和抗体的能力。
gEdelta3.1IBR病毒在病毒gE基因编码序列中存在缺失，其中插入了一个稳定的嵌合报告基因（β - gal）。重组病毒和亲本病毒的一步生长动力学和病毒产量相似，但在感染早期，重组病毒产量相对较低。经鼻内接种后，重组gEdelta3.1IBR病毒在犊牛上呼吸道中复制，但产生的子代病毒数量减少数百倍，病毒排出持续时间较短。犊牛体内实验和血清中和试验结果表明，缺失gE基因对诱导抗BHV - 1中和抗体影响不大，但足以降低BHV - 1在犊牛中的毒力。
