弓形虫表面主要抗原1(SAG1)蛋白的原核表达与鉴定

Prokaryotic expression and identification of major surface antigen 1(SAG1)protein of Toxoplasma gondii

为了实现弓形虫主要表面抗原1(surface antigen 1,SAG1)蛋白在原核表达系统中的高效表达,试验根据GenBank中弓形虫SAG1基因序列(登录号为S76248.1)及大肠杆菌密码子偏好性对目的基因序列进行优化设计,以pET-32a(+)为表达载体构建重组表达质粒pET32a(+)-SAG1,并将其转化至大肠杆菌BL21(DE3)感受态细胞中,用异丙基-β-D-硫代吡喃半乳糖苷(IPTG)对重组菌进行诱导,表达重组蛋白,通过优化诱导条件实现对重组蛋白的大量表达,使用NGC^(TM)层析纯化系统纯化重组蛋白,并分别以抗His标签单克隆抗体和犬弓形虫阳性血清作为一抗、羊抗鼠IgG-HRP和HRP-兔抗犬作为二抗对重组蛋白进行Western-blot鉴定。结果表明:重组表达质粒pET32a(+)-SAG1经双酶切分别在5 900 bp和1 020 bp处出现1条载体条带和1条目的基因条带,与预期结果相符;重组蛋白大小为55 ku且以包涵体的形式成功表达,当诱导条件为25℃、0.4 mmol/L IPTG诱导8 h时重组蛋白的表达量最高;纯化后的重组蛋白条带单一,纯度较高,3个样品质量浓度分别为0.409 5,0.368 5,0.451 9 mg/mL;表达的重组蛋白与抗His标签单克隆抗体和犬弓形虫阳性血清均能特异性结合。说明在大肠杆菌中成功表达了弓形虫SAG1重组蛋白,纯化后的重组蛋白纯度和质量浓度较高,且具有良好的反应原性。

In order to realize the efficient expression of Toxoplasma gondii major surface antigen 1(SAG1)protein in the prokaryotic expression system,in the experiment,according to the Toxoplasma gondii SAG1 gene sequence in GenBank(accession number S76248.1)and codon preference of E.coli,the target gene sequence was optimally designed.The recombinant expression plasmid pET32a(+)-SAG1 was constructed with pET-32a(+)as expression vector and transformed into E.coli BL21(DE3)competent cells;the recombinant bacteria were induced with isopropyl-β-D-thiogalactopyranoside(IPTG)to express the recombinant protein.Large amount of recombinant protein was expressed by optimizing the induction conditions;the recombinant protein was purified by the NGC^(TM) chromatographic purification system.And the recombinant protein was identified by Western-blot using monoclonal anti-HIS tag antibody and Toxoplasma gondii positive serum in dogs as primary antibody respectively,and sheep anti-mouse IgG-HRP and HRP-rabbit anti-dog as secondary antibody.The results showed that the recombinant expression plasmid pET32a(+)-SAG1 showed one vector band and one target gene band at 5900 bp and 1020 bp,respectively,which were consistent with the expected results.The recombinant protein was 55 ku in size and was successfully expressed as inclusion bodies,and the highest expression of recombinant protein was achieved when the induction conditions were 25℃and 0.4 mmol/L IPTG for 8 h.The purified recombinant protein had a single band and high purity;the mass concentrations of the three samples were 0.4095,0.3685,and 0.4519 mg/mL,respectively.The expressed recombinant protein specifically bound to both anti-HIS tag monoclonal antibody and Toxoplasma gondii positive serum in dogs.The results suggested that Toxoplasma gondii SAG1 recombinant protein was successfully expressed in E.coli,and the purified recombinant protein had high purity and mass concentration with good reactivity.