热休克蛋白gp96（又称GRP94）是位于细胞内质网膜上的热休克蛋白90家族中的一员，在天然免疫和获得性免疫中发挥着重要作用。gp96能作为分子伴侣结合细胞中的肿瘤抗原、病毒抗原或胞内细菌抗原，并将结合的抗原表位呈递给抗原递呈细胞的“专用运输车”MHCI类和II类分子，从而启动特异性T细胞CD8+和CD4+T细胞免疫应答。另一方面，gp96分子本身作为免疫活性分子通过与Toll样受体（TLR）作用，促进CD8+和CD4+T细胞的增殖和分泌细胞因子，促进抗原呈递细胞成熟并增强其功能。
中科院微生物研究所的病原微生物研究团队自1997年开展gp96免疫学功能的研究，探索gp96在乙肝感染中介导的T细胞免疫，取得了多项重要进展。2011年在前期研究中发现调节性T细胞（Treg）对gp96免疫活性起负调控作用，课题组进一步查明清除Treg可显著提高gp96的T细胞免疫活性和抗肿瘤活性，为提高gp96疫苗的免疫活性以及目前gp96作为肿瘤自体疫苗在临床中治疗肿瘤提供理论指导；通过乙肝病毒（HBV）转基因鼠模型，发现gp96介导的T细胞免疫应答可有效抑制和清除乙肝病毒，gp96佐剂疫苗能有效激活HBV特异性T细胞，并降低转基因鼠Treg的数量。小鼠免疫5-8周后血清中的HBVS抗原降低50%以上，病毒DNA拷贝数下降1000倍，肝脏细胞中病毒清除95%以上，表明gp96治疗性疫苗免疫能有效打破免疫耐受、激活T细胞清除病毒，这为研发gp96乙肝治疗性疫苗提供依据；同时，首次应用汉逊酵母表达出有免疫活性的重组gp96蛋白，为研究gp96的生物学功能和疫苗佐剂开发奠定了基础。
以上研究分别发表在Cancer Immunology Immuno therapy，Vaccine和《生物技术期刊》（Journal of Biotechnology），并申请了2项中国发明**。（生物谷 Bioon.com）
doi:10.1016/j.jbiotec.2010.12.006
PMC:
PMID:
Hansenula polymorpha expressed heat shock protein gp96 exerts potent T cell activation activity as an adjuvant.
Li Yang Y,Song Haolei H,Li Jin J,Wang Yanzhong Y,Yan Xiaoli X,Zhao Bao B,Zhang Xiaojun X,Wang Saifeng S,Chen Lizhao L,Qiu Bingsheng B,Meng Songdong S,
Previous studies together with ours showed that heat shock protein gp96 as an adjuvant induces antigen specific T cell responses against cancer and infectious diseases. However, at present there is no efficient method to obtain high amount of full-length gp96 by in vitro expression. Here, we used the yeast Hansenula polymorpha as an efficient host for gp96 recombinant protein production. The transformant clones with highly expressed recombinant proteins were screened and selected by measuring the halo size which indicates enzymatic hydrolysis of starch in the medium. High-level production of gp96 (around 150mg/mL) was achieved by using high-cell density fed-batch cultivations. We showed that peptide binding of the recombinant protein has similar specificity and intrinsic binding parameters as that of the native gp96. We next examined the self-assembly properties and high-order structures of the recombinant protein. Moreover, the H. polymorpha expressed recombinant gp96 can effectively induce HBV-specific CTL response in immunized mice while Escherichia coli-expressed gp96 cannot. Our results therefore may provide bases for structure and functional studies of gp96 and thereby potentially expedite the development of gp96-based vaccines for immunotherapy of cancer or infectious diseases.