利用多基因缺失型杆状病毒在昆虫细胞中生产腺相关病毒

腺相关病毒(adeno-associated virus, AAV)是基因治疗领域最常使用的病毒载体之一,产量低、成本高是该产业面临的关键瓶颈问题。本研究旨在基于多基因缺失型杆状病毒,建立双病毒感染昆虫细胞以生产AAV的技术体系。首先,进行AAV生产用多基因缺失型重组杆状病毒的构建和扩增,并检测杆状病毒滴度及其感染细胞的效果;然后,使用双杆状病毒共感染昆虫细胞,并优化感染条件;最后,基于优化条件进行AAV生产,并检测评估产量、质量等相关指标。结果表明,AAV生产用多基因缺失型杆状病毒滴度较野生型无差异,感染后细胞存活率下降明显减缓。使用双病毒路线进行AAV优化生产,Bac4.0-1的基因组滴度为1.63×10~(11)VG/mL,Bac5.0-2的基因组滴度为1.02×10~(11) VG/mL,较野生型产量分别提升了240%和110%。电镜下,3组均具有正常的AAV病毒形态,且转导活性相近。本研究建立了基于多基因缺失型杆状病毒感染昆虫细胞的AAV生产体系,显著提高了AAV产量,具有一定的应用价值。

双向选择标记amdS在黑曲霉中自发突变及amdS-检测

目的将传统上作为营养缺陷标记的amdS进行诱变,使其成为双向选择标记,有利于构建黑曲霉多基因缺失菌株。方法以已有amdS作选择标记的黑曲霉双基因缺失株为出发突变株,在以尿素为唯一氮源的氟代乙酰氨培养基上筛选amdS-突变菌株。结果将另一以amdS作选择标记的基因进行转化,得到一株三基因缺失菌株。结论在黑曲霉中利用amdS可作为双向选择标记的特点,构建多基因缺失菌株是行之有效的。

Lack of association between ADRA2B-4825 gene insertion/deletion poly- morphism and migraine in Chinese Han population

Objective The present study aimed to estimate the association between susceptibility to migraine and the 12nucleotide insertion/deletion （indel） polymorphism in promoter region ofα 2B -adrenergic receptor gene （ADRA2B）.Methods A case-control study was carried out in Chinese Han population,including 368 cases of migraine and 517 controls.Genomic DNA was extracted from blood samples,and DNA fragments containing the site of polymorphism were amplified by PCR.Data were adjusted for sex,age,migraine history and family history,and analyzed using a logistic regression model.Results There was no association between indel polymorphism and migraine,at either the allele or the genotype level.Conclusion These findings do not support a functional significance of ADRA2B indel polymorphism at position-4825 relative to the start codon in the far upstream region of the promoter in the present migraine subjects.