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李大金, Da-Jin Li*, Hong-Mei Wang, Lei Li, Xin-Rong Zhao, Ming-Yan Wang, Ying Zhu, Yi Meng, Min-Min Yuan
Journal of Reproductive Immunology 60(2003)129-141,-0001,():
-1年11月30日
Objective: To express the hCGβ-C3d3 fusion protein in a CHO cell continual expression system to investigate further the adjuvant effects of C3d on contraceptive vaccination. Method: We constructed a plasmid pcDNA3-hCGβ-C3d3 which contains three copies of murine C3d cDNA and the hCGβ gene by cloning the chimerical hCGβ-C3d3 cDNA into the eukaryotic vector pcDNA3 downstream of the CMV promoter. The plasmid was transfected into a COS-7 cell transient expression system and a CHO cell continual expression system. RIA was used to detect hCGβ in the culture supernatant. Western blot and Raji cell immunohistochemical assays were performed to evaluate the expressed protein. Then, 6-/8-week-old female BALB/c mice were inoculated intramuscularly with pcDNA3-hCGβ and pcDNA3-hCGβ-C3d3, and ELISA was used to assess anti-hCGβ IgG antibody in serum. Results: In 72 h after COS-7 cells were transfected with the plasmid pcDNA3-hCGβ-C3d3, 1.0/105 cells could secrete 152 ng of the recombinant protein (calculated by hCGβ contained). The transfected CHO cells, which were then screened by G418, could continuously secrete the fusion protein at 660 ng/106 cells/48 h. The hCGβ-C3d3 protein was purified by anti-hCGβ immunoaffinity chromatography. Raji cell immunohistochemical assay demonstrated that both the hCGβ and C3d3 were successfully fused. After DNA immunization intramuscularly, the anti-hCGβ IgG antibody titer in the pcDNA3-hCGβ-C3d3 immunized group was 243-fold higher than that of the pcDNA3-hCGβ immunized group. Conclusion: We have expressed the hCGβ-C3d3 protein successfully, both in a transient expression system (COS-7 cells) and in a stable expression system (CHO cells). The C3d3 molecular adjuvant can enhance significantly the immunogenecity of hCGβ antigen in DNA immunization.
hCGβ, C3d molecular adjuvant, Expression in vitro, Eukaryotic cell, DNA immunization
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【期刊论文】The Expression of CXCR4/CXCL12 in First-Trimester Human Trophoblast Cells1
李大金, Xia Wu, Da-Jin Li, Min-Min Yuan, Ying Zhu, and Ming-Yan Wang
BIOLOGY OF REPRODUCTION 70, 1877-1885 (2004),-0001,():
-1年11月30日
Chemokines and chemokine receptors have been implicated as pivotal players in many physiological and pathological situations, but little is known about the expression and function of chemokines and chemokine receptors at the materno-fetal interface. In this study, we first analyzed the transcription of 18 chemokine receptors in first-trimester human trophoblast cells. Among these receptors, CXCR4 was found highly transcribed. We demonstrated afterward that both CXCR4 and CXCL12 (stromal cell-derived factor-1; SDF-1) were expressed in trophoblast cells. Primary cultured trophoblast cells were also found secreting CXCL12 spontaneously. To identify the functional role of CXCR4/CXCL12 in these cells, we treated trophoblast cells with recombinant human (rh) SDF-1a and analyzed the cell viability and signaling pathway. The results showed that rhSDF-1a increased the viability of trophoblast cells and the activation of extracellular signal-regulated kinases signaling pathway in vitro. Our findings suggest that first-trimester trophoblast cells express functional CXCR4/CXCL12, which may play an important role in early pregnancy such as stimulating trophoblast cell proliferation or differentiation in an autocrine manner.
Cytokines, trophoblast
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李大金, Li-Ping Jin, *, Da-Jin Li, Jin-Ping Zhang, †, Ming-Yan Wang, Xiao-Yong Zhu, Ying Zhu, Yi Meng, * and Min-Min Yuan*
The Journal of Immunology, 2004, 173: 3612-3619.,-0001,():
-1年11月30日
The embryo expresses paternal Ags foreign to the mother and therefore has been viewed as an allograft. It has been shown that anergic T cells generated by blocking of the CD28/B7 costimulatory pathway with anti B7-1 and anti B7-2 mAbs can be transferred as suppresser cells to prevent allograft rejection. Little is known, however, about the in vivo function of anti-B7-treated T cells after their transfer into abortion-prone mice in the maintenance of materno-fetal tolerance. In the present study, abortion-prone CBA/J females mated with DBA/2 males were administered anti-B7-1 and anti-B7-2 mAbs on day 4 of gestation (murine implantation window). The anti-B7-treated T cells subsequently were adoptively transferred into abortion-prone CBA/J mice. We demonstrated that costimulation blockade with anti-B7 mAbs at the time of implantation resulted in altered allogeneic T cell response and overcame increased maternal rejection to the fetus in the CBA/J DBA/2 system. The transferred anti-B7-treated T cells appeared to be regulatory, decreasing responsiveness and generating clonal deviation in maternal recipient T cells. The transferred CFSE-labeled T cells were found to reside in the spleen and uterine draining lymph nodes, and a few were localized to the materno-fetal interface of the maternal recipient. Our findings suggest that the anti-B7-treated T cells not only function as potent suppresser cells, but also exert an immunoregulatory effect on the maternal recipient T cells, which cosuppresses maternal rejection to the fetus. This procedure might be considered potentially useful for fetal survival when used as an immunotherapy for human recurrent spontaneous abortion.
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李大金, Xiu-Li Wang, Da-Jin Li*, Min-Min Yuan, Min Yu, Xiao-Ying Yao
Journal of Reproductive Immunology 63(2004)97-110,-0001,():
-1年11月30日
The vaccine directed against human chorionic gonadotropin (hCG) has previously undergone clinical test demonstrating the feasibility of the approach in preventing pregnancy in women. Some individuals, however, did not response adequately despite employing highly immunogenic bacterial toxoids as carriers. In this study, we investigated the potential of three copies of C3d as a new molecular adjuvant to enhance the immunogenicity of hCGβ protein antigen. The antibody response to the hCGβ-C3d3 fusion protein immunization was compared with those resulting from immunization with the hCGβ alone and the hCGβ plus CFA/IFA either in BALB/c mice or in C57BL/6J mice. Our results showed that the fusion of C3d3 to hCGβ protein antigen resulted in a significant elevation of the serum anti-hCGβ antibody level in the two mouse strains and the antibodies were capable of effectively neutralizing the bioactivity of hCG. The immunization with C3d3 as a molecular adjuvant favored Th2 bias of immune response. The immunity-enhancing effect of the C3d3 was 10-fold (initial) and 20–32-fold (booster) greater than CFA/IFA. These findings indicated that fusion of C3d3 to hCGβ, as a means of harnessing the adjuvant potential of the innate immune system, may improve immunogenicity of the hCGβ contraceptive vaccine, which is useful to produce a cost-effective vaccine and for the less-responsive population.
hCGβ, Protein antigen, C3d3, Molecular adjuvant, Humoral immunity
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李大金, Xiao-Ying Yao, Hong-Mei Wang, Da-Jin Li*, Min-Min Yuan, Xiu-Li Wang, Min Yu, Ming-Yan Wang, Ying Zhu, Yi Meng
Journal of Reproductive Immunology 63(2004)111-122,-0001,():
-1年11月30日
Objectives: To test the possibility of vaccination with lactobacillus expressing hCGβ antigen administered by vaginal mucosal immunization. Methods: A plasmid pIlac-hCGβ was constructed and then transfected into Lactobacillus casei CECT5276, which stably expressed hCGβ protein. RIA was used to detect hCGβ in the culture supernatant and cell lysate.Western blotting was performed to evaluate the expressed protein of interest. Female BALB/c mice aged 6–8 weeks received inoculations in the vagina of the recombinant L. casei CECT5276. ELISA was used to determine the anti-hCGβ IgA antibody in vaginal lavage fluid from the BALB/c mice after vaginal mucosal immunization. Results: The pIlac alone appeared to have a higher efficiency than pIlac-hCGβ, and the highest transfection efficiency of both plasmids was at pulse voltages of 1200V and 1500V. About 78.5% of the hCGβ protein was excreted into the culture supernatant. Excretion of hCGβ was most efficient when the pH of the culture medium was adjusted to around 7.0 and the concentration of lactose was around 1%. The hCG protein in the vaginal lavage fluid of these BALB/c mice was positive on the third day after vaginal inoculation. Anti-hCGβ IgA antibody continued to be found in the vaginal lavage fluid for 2 weeks following a booster vaginal inoculation. The splenic lymphocytes of the mice immunized with hCGβthrough the vagina underwent a proliferative reaction to hCGβ antigen restimulation in vitro. Interferon gamma (IFN-γ) and interleukin (IL)-4 were secreted at higher levels after vaginal mucosal immunization of L. casei expressing hCG than after vaginal mucosal immunization of L. casei alone. Conclusions: Vaginal immunization of lactobacillus expressing hCG induced an antihCGβ antibody response in the murine vaginal mucosa. Induction of the antigen-specific antibodies in the reproductive tract following vaginal inoculation of recombinant lactobacillus will lead to the development of a safe, efficient, and easy-to-use form of immunocontraception.
Recombinant lactobacillus, Immunocontraception, hCGβ, Reproductive tract, Mucosal immunization
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