Jiangsu Agri-animal Husbandry Vocational College, Veterinary Bio-pharmaceutical, Jiangsu Key Laboratory for High-Tech Research and Development of Veterinary Biopharmaceuticals, Taizhou, 225300, China.
Poult Sci. 2017 Jul 1;96(7):2447-2453. doi: 10.3382/ps/pex029.
Due to its antimicrobial properties and low toxicity, human lysozyme (hLYZ) has broad application in the medical field and as a preservative used by the food industry. However, limited availability hinders its widespread use. Hence, we constructed a recombinant avian adeno-associated virus (rAAAV) that would specifically express hLYZ in the chicken oviduct and harvested hLYZ from the egg whites of laying hens. The oviduct-specific human lysozyme expression cassette flanked by avian adeno-associated virus (AAAV) inverted terminal repeats (ITRs) was subcloned into the modified baculovirus transfer vector pFBX, and then the recombinant baculovirus rBac-ITRLYZ was generated. The recombinant avian adeno-associated virus was produced by co-infecting Sf9 cells with rBac-ITRLYZ and the other 2 baculoviruses containing AAAV functional genes and structural genes, respectively. Electron microscopy and real-time PCR revealed that the recombinant viral particles were generated successfully with a typical AAAV morphology and a high titer. After one intravenous injection of each laying hen with 2 × 1011 viral particles, oviduct-specific expression of recombinant human lysozyme (rhLYZ) was detected by reverse transcription-PCR. The expression level of rhLYZ in the first wk increased to 258 ± 11.5 μg/mL, reached a maximum of 683 ± 16.4 μg/mL at the fifth wk, and then progressively declined during the succeeding 7 wk of the study. Western blotting indicated that the oviduct-expressed rhLYZ had the same molecular weight as the natural enzyme. These results indicate that an efficient and convenient oviduct bioreactor mediated by rAAAV has been established, and it is useful for production of other recombinant proteins.
由于其抗菌特性和低毒性，人溶菌酶（hLYZ）在医学领域和食品工业防腐剂中有广泛的应用。然而，其有限的可用性限制了它的广泛应用。因此，我们构建了一种重组禽腺相关病毒（rAAAV），该病毒可以特异性地在鸡输卵管中表达 hLYZ，并从产蛋鸡的蛋清中提取 hLYZ。侧翼带有禽腺相关病毒（AAAV）反向末端重复序列（ITR）的输卵管特异性人溶菌酶表达盒被亚克隆到修饰的杆状病毒转移载体 pFBX 中，然后生成重组杆状病毒 rBac-ITRLYZ。通过共感染 Sf9 细胞，用 rBac-ITRLYZ 和另外两个分别含有 AAAV 功能基因和结构基因的杆状病毒，产生了重组禽腺相关病毒。电子显微镜和实时 PCR 显示，成功地产生了具有典型 AAAV 形态和高滴度的重组病毒颗粒。在每个产蛋鸡静脉注射 2×1011 个病毒颗粒后，通过反转录-PCR 检测到输卵管特异性表达重组人溶菌酶（rhLYZ）。rhLYZ 的表达水平在第 1 周增加到 258±11.5μg/mL，在第 5 周达到最大值 683±16.4μg/mL，然后在研究的接下来的 7 周内逐渐下降。Western blot 表明，输卵管表达的 rhLYZ 与天然酶具有相同的分子量。这些结果表明，已经建立了一种高效、便捷的 rAAAV 介导的输卵管生物反应器，可用于生产其他重组蛋白。
