原核表达人41型腺病毒(Ad41) 蛋白 V及其抗血清的制备

Preparation of antiserum to protein V of human adenovirus type 41

摘要:

摘要：目的 人肠腺病毒Ad41被称为难养腺病毒，其难以培养的特性可能与次要核心蛋白V（Ad41 protein V，pV）表达不充分有关。本研究拟表达纯化Ad41 pV抗原，免疫动物制备抗血清，为研究Ad41难养性机理打下基础。方法 以野生型Ad41基因组DNA为模板，PCR扩增pV，克隆到原核表达载体pET30a(+)，测序后，转化大肠杆菌BL21(DE3)菌株，异丙基-β-D-硫代半乳糖苷（IPTG）诱导目的蛋白表达，固化金属亲和层析(IMAC)方法纯化，免疫BALB/c小鼠制备抗血清，将获得的抗血清用于Western blot检测Ad41感染各细胞系后pV的表达。结果 克隆得到包括完全编码区的pV基因，表达质粒转化BL21(DE3)菌株，使用1 m mol/L IPTG 37℃诱导4 h，pV以包涵体形式表达，或使用0.5 m mol/L IPTG 25℃诱导8 h获得可溶性表达。利用皮下多点注射包涵体的方法免疫小鼠，得到抗pV抗血清；使用纯化的可溶性pV作为抗原对抗血清进行了鉴定，表明该抗血清可用于Western blot检测。等量野生型Ad41感染293或293E12细胞（一株稳定表达Ad41 E1B55K基因的293细胞）后，pV在293E12细胞的表达明显高于293细胞。结论 成功克隆了Ad41 pV基因，表达纯化了重组蛋白，获得了可用于Western blot检测的抗血清，为进一步研究Ad41难养性机理打下了基础。

Abstract:

Abstract: [Objective] The fastidious property of human adenovirus type 41 (Ad41) may be resulted from inadequate expression of protein V (pV), the minor core protein of adenovirus, in packaging cells. In this report, we prepared antiserum to pV of Ad41 and studied the mechanism of Ad41 fastidiousness. [Methods] Coding sequence of pV was amplified by PCR with the genome DNA of wild Ad41 (NIVD103) as template, and cloned into pET30a(+) vector to generate a recombinant plasmid called pET-pV. His-tag-fused pV was expressed in pET-pV-transformed E. Coli strain BL21(DE3) by adding the inducer of Isopropy β-D-1-Thiogalactopyranoside (IPTG) and purified with the method of immobilized metal ion affinity chromatography (IMAC). Antiserums to pV were collected from pV inclusion bodies-immunized mice and evaluated by Western blot. [Results] The sequencing assay showed that the cloned pV gene was highly homologous with that of Ad41 Tak strain, and there were only three residues changed in the corresponding amino-acid sequence. pV was expressed as inclusion bodies or in soluble form in BL21(DE3) cells under inducing condition of 1 m mol/L IPTG, 37 °C, 4 h or 0.5 m mol/L IPTG, 25 °C, 8 h, respectively. Antiserums to pV from most immunized mice were highly effective for Western blot assay. After infected with equivalent Ad41, 293E12, an Ad41 E1B55K-transfected 293 cell line, expressed more pV than 293 cells. [Conclusion] We successfully prepared antiserums to Ad41 pV and it could be used in Western blot assay to study the fastidious property of Ad41.