Characterization of the cellular immune response induced by Mycobacterium tuberculosis Rv2626c / 生物工程学报

Nearly a quarter of the world's population is infected with Mycobacterium tuberculosis and remains long-term asymptomatic infection. Rv2626c is a latent infection-related protein regulated by DosR of M. tuberculosis. In this study, the Rv2626c protein was prokaryotically expressed and purified, and its immunobiological characteristics were analyzed using RAW264.7 cells and mice as infection models. SDS-PAGE and Western blotting analysis showed that the Rv2626c-His fusion protein was mainly expressed in soluble form and specifically reacted with the rabbit anti-H37RV polyclonal serum. In addition, we found that the Rv2626c protein bound to the surface of RAW264.7 macrophages and up-regulated the production of NO. Moreover, the Rv2626c protein significantly induced the production of pro-inflammatory cytokines IFN-γ, TNF-α, IL-6 and MCP-1, and induced strong Th1-tendency immune response. These results may help to reveal the pathogenic mechanism of M. tuberculosis and facilitate the development of new tuberculosis vaccine.

Construction and characterization of an attenuated recombinant Listeria monocytogenes vector vaccine delivering HPV16 E7 / 生物工程学报

Listeria monocytogenes (L. monocytogenes, LM) is an excellent tumor vaccine vector. In this study, recombinant LM vaccine candidate expressing human papillomavirus type 16 (HPV16) E7 protein was constructed and its charactericts were determined. Through homologous recombination, E7 gene was cloned in frame with the LM4 Phly promoter-signal sequence, and introduced into the chromosome of LM4. The recombinant strain named LM4△hly::E7 with the plasmid-free and antibiotic-resistant gene-free was constructed. LM4△hly::E7 could express and secrete E7-LLO fusion protein; its size is 66 kDa and has immunological activity. Furthermore, LM4△hly::E7 could multiply in RAW264.7 macrophages by confocal laser scanning microscope. Additionally, LM4△hly::E7 could induce specific antibodies against E7 in immunized mice in ELISA. Also, the 50% lethal dose (LD₅₀) of LM4△hly::E7 strain was 3.863×10⁹ CFU (Colony-Forming Units) in C57BL/6 mice with intraperitoneal immunization, which was more attenuated than wild type LM4. Mice immunized with LM4△hly::E7 did not show obvious pathological change. These data show that LM4△hly::E7 expressing E7-LLO fusion protein has good safety, which may provide the materials for research of antitumor effect and would be a promising vaccine candidate for cervical cancer.

Isolation of the intracellular and extracellular polysaccharides of Ganoderma neojaponicum (Imazeki) and characterization of their immunomodulatory properties

Background The role of polysaccharides isolated from the Ganoderma species of fungi in innate immunity has recently become a topic of research. Although some work has been conducted concerning Ganoderma lucidum, the characteristics of polysaccharides isolated from Ganoderma neojaponicum (Imazeki) as immunomodulatory agents are largely unknown. The aims for this study were to isolate and characterize the intracellular polysaccharides (IPSs) and extracellular polysaccharides (EPSs) of G. neojaponicum from STR reactor. Results The production of EPS and IPS was optimized on day 4 of the cultivation time in 2 L STR reactor based on the amount of biomass yield, total carbohydrate, β-glucan and a-glucan content. Further analysis, both the EPSs and IPSs showed the enhancement on proliferation and increment of phagocytosis activities of macrophage (RAW264.7) cell lines. Using an oral toxicity test, we also observed that 2000 mg/kg body weight/day dosage of dried G. neojaponicum mycelium does not cause any significant toxic effects on Sprague-Dawley rats in 14 d of administration. Conclusion The findings of this study indicate that the IPSs and EPSs of G. neojaponicum have the potential to be used as immunomodulating agents to stimulate the innate immune system for fighting infectious diseases. The polysaccharides from G. neojaponicum have to be further commercially explored as an alternative for medicinal Ganoderma variety of G. lucidum production.

Repressive effects of RNA interference technique on expression and function of glucocorticoid receptor in human macrophage cell line U937 / 第三军医大学学报

Objective To establish a glucocorticoid receptor knockdown model of human macrophage cell line U937 with RNA interference technique. Methods Two RNAi recombinant plasmids (named pSilencer 3.1-GR 1 and pSilencer 3.1-GR 2) targeting to GR gene were constructed. After RNAi recombinant plasmids were transfected into human macrophage cell line U937, the expressions of GR mRNA and GR protein were evaluated with RT-PCR and Western blotting respectively. The transcriptional activation function of GR was evaluated through the detection of relative luciferase activity after dexamethasone treatment. Results Two RNAi recombinant expression plamids were constructed and identified by sequencing. pSilencer 3.1-GR 2 transfection could inhibit not only GR mRNA and protein expressions, but also transcriptional activation function of GR specially; pSilencer 3.1-GR 1 transfection had no significant changes as compared to normal control. Conclusion A glucocorticoid receptor knockdown model has been established successfully, which offers a new method for the further research of GR biological functions.