Feng Gaifeng, Jin Hui, Wang Weixi, Qian Yihua, Wang Quanying, Yang Guangxiao
Department of Human Anatomy and Histo/Embryology, School of Medicine, Xi'an Jiaotong University, Xi'an 710061,China.
Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2012 Mar;37(3):290-5. doi: 10.3969/j.issn.1672-7347.2012.03.014.
To construct a recombinant prokaryoticexpression plasmid pET/ c-Aβ(15)-c, and evaluate the immunogenicity of its encoded fusion protein as expressed in E.coli.
The gene fragment HBc88-144 was amplified by PCR and subcloned to pUC19. The synthetic, double-strand Aβ(1-15) gene was inserted downstream of HBc1-71 in pGEMEX/c1-71. After restriction enzyme digestion, c1-71- Aβ(15) was spliced to HBc88-144, yielding the recombinant gene c-Aβ(15)-c; that gene was subcloned into pET-28a(+). The fusion protein (CA15C) expressed in the transformed E.coli BL21 was induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) and analyzed by SDS-PAGE. The virus-like particle (VLP) formed by fusion protein CA15C was observed with transmission electric microscope (TEM). Four Kunming (KM) mice were given intraperitoneal injections of CA15C, and the anti-Aβ antibody elicited was detected by indirect ELISA.
The sequence of the recombinant gene was confirmed by restriction enzyme digestion and DNA sequencing. After IPTG induction, the fusion protein was expressed, mainly in the sediment from the bacterial lysate. The expression level was 40% of total protein in the sediment. The CA15C could form VLP. After 5 rounds of immunization, the titer of anti-Aβ antibody in the sera of KM mice reached 1:10000, while the anti-HBc antibody was undetectable.
Recombinant c-Aβ(15)-c gene can be expressed in E.coli. The expressed protein can form VLPs and has a strong immunogenicity.
构建重组原核表达质粒pET/c-Aβ(15)-c，并评价其在大肠杆菌中表达的编码融合蛋白的免疫原性。
通过PCR扩增基因片段HBc88-144，并亚克隆至pUC19。将合成的双链Aβ(1-15)基因插入pGEMEX/c1-71中HBc1-71的下游。经限制性内切酶消化后，将c1-71-Aβ(15)与HBc88-144拼接，得到重组基因c-Aβ(15)-c；将该基因亚克隆至pET-28a(+)。用异丙基β-D-1-硫代半乳糖苷（IPTG）诱导转化的大肠杆菌BL21中表达的融合蛋白（CA15C），并通过SDS-PAGE进行分析。用透射电子显微镜（TEM）观察融合蛋白CA15C形成的病毒样颗粒（VLP）。对4只昆明（KM）小鼠腹腔注射CA15C，通过间接ELISA检测产生的抗Aβ抗体。
通过限制性内切酶消化和DNA测序确认重组基因序列。IPTG诱导后，融合蛋白表达，主要存在于细菌裂解物的沉淀中。表达水平占沉淀中总蛋白的40%。CA15C可形成VLP。5轮免疫后，KM小鼠血清中抗Aβ抗体效价达到1:10000，而抗HBc抗体未检测到。
重组c-Aβ(15)-c基因可在大肠杆菌中表达。表达的蛋白可形成VLPs，具有较强的免疫原性。
