Human thymic stromal lymphopoietin promotes the proliferation and invasion of cervical cancer cells by downregulating microRNA-132 expression.

Thymic stromal lymphopoietin (TSLP), produced by cervical cancer (CC) cells, promotes angiogenesis, and the recruitment and functional regulation of eosinophils. It has been reported that microRNA (miR)-132 is aberrantly decreased in CC tissues. However, the function and mechanism of TSLP on the biological behaviors of CC cells is largely unknown. The aim of the present study was to investigate the effect of TSLP on the expression of miR-132 and the proliferation and invasion in vitro of CC cell lines, namely, HeLa and SiHa cells. The transcrpitional level of miR-132 was analyzed using reverse transcription-quantitative polymerase chaon reaction. The proliferation, invasion, and the expression of proliferation and invasion-related molecules in HeLa and SiHa cells in vitro were evaluated using bromodeoxyuridine cell proliferation, Matrigel invasion assays, flow cytometry and ELISA, respectively. Here, it was revealed that recombinant human TSLP (rhTSLP) downregulated the expression levels of miR-132 in HeLa and SiHa cells, and by contrast, the neutralizing antibodies for TSLP or TSLP receptor (TSLPR) upregulated miR-132 expression levels in HeLa and SiHa cells. The overexpression of miR-132 resulted in a lowered proliferation and invasiveness, decreased levels of proliferation-associated molecules marker of proliferation Ki-67 and proliferating cell nuclear antigen, and the decreased production of matrix metalloproteinase (MMP)2 and MMP9 in HeLa and SiHa cells. Compared with the control group, there was a higher level of proliferation and invasion in HeLa and SiHa cells following stimulation with rhTSLP. However, these effects induced by rhTSLP were significantly impaired in HeLa and SiHa cells with miR-132 overexpression. The results of the present study indicated that TSLP produced by CC cells downregulated miR-132 expression, and stimulated the proliferation and invasion of CC cells, thereby further promoting the development of CC.

Cisplatin inhibits the growth, migration and invasion of cervical cancer cells by down-regulating IL-17E/IL-17RB.

Interleukin (IL)-17E mainly produced by immune cells, is a distinct member of the IL-17 cytokine family, which has multifarious immunomodulatory activities. As a potent anticancer drug, cisplatin is commonly used against various types of solid tumors. The present study was performed to investigate whether cisplatin regulates the expression of IL-17E and it receptor IL-17RB, and the role of IL17E in cervical cancer cells in vitro. The expression of IL-17E and IL-17RB in cervical cancer cells was detected by flow cytometry and ELISA. The viability, apoptosis, migration and invasion of cervical cancer cells were analyzed by CCK8, Annexin V-7AAD apoptosis, transwell migration, wound healing, and matrigel invasion assays. Here, we found that cervical cancer cells co-expressed IL-17E and IL-17RB, especially HeLa and SiHa cells. Recombinant human IL-17E protein (rhIL-17E) enhanced the viability, migration and invasion of HeLa and SiHa cells, and blocking IL-17E with anti-human IL-17RE neutralizing antibody promoted the apoptosis of HeLa and SiHa cells. Cisplatin significantly down-regulated the expression of IL-17E and IL-17RB, and further reversed the regulatory effects of rhIL-17E on viability, apoptosis, migration and invasion of HeLa and SiHa cells. The results suggest that cisplatin inhibits the viability, migration, invasion, and promotes the apoptosis of cervical cancer cells possibly by down-regulating IL-17E/17RB signaling. Cisplatin may be the first choice for cervical cancer patients with abnormally high IL-17E expression.

Chemokine CCL17 induced by hypoxia promotes the proliferation of cervical cancer cell.

Cervical cancer is often associated with hypoxia and many kinds of chemokines. But the relationship and role of hypoxia and Chemokine (C-C motif) ligand 17 (CCL17) in cervical cancer are still unknown. Here, we found that CCL17 was high expressed in cervical cancer. HeLa and SiHa cells could secrete CCL17 in a time-dependent manner. Hypoxia increased expression of CCL17 receptor (CCR4) on HeLa and SiHa cells. Treatment with recombination human CCL17 (rhCCL17) led to an elevation of cell proliferation in HeLa and SiHa cells in a dose-dependent manner. In contrast, blocking CCL17 with anti-human CCL17 neutralizing antibody (α-CCL17) played an oppose effect. However, rhCCL17 had no effect on apoptosis in cervical cancer cells. Further analysis showed that hypoxia promoted the proliferation of HeLa and SiHa cells, and these effects could be reversed by α-CCL17. Stimulation with the inhibitor for c-Jun N-terminal kinase (JNK) or signal transducers and activator of transcription 5 (STAT5) signal pathway not only directly decreased the proliferation of HeLa and SiHa cells, but also abrogated the stimulatory effect of rhCCL17 on the proliferation of HeLa and SiHa cells. These results suggest that a high level of CCL17 in cervical cancer lesions is an important regulator in the proliferation of cervical cancer cells through JNK and STAT5 signaling pathways. In this process, hypoxia magnifies this effect by up-regulating CCR4 expression and strengthening the interaction of CCL17/CCR4.

Chemokine CCL24 promotes the growth and invasiveness of trophoblasts through ERK1/2 and PI3K signaling pathways in human early pregnancy.

Chemokine CCL24, acting through receptor CCR3, is a potent chemoattractant for eosinophil in allergic diseases and parasitic infections. We recently reported that CCL24 and CCR3 are co-expressed by trophoblasts in human early pregnant uterus. Here we prove with evidence that steroid hormones estradiol (E), progesterone (P), and human chorionic gonadotropin (hCG), as well as decidual stromal cells (DSCs) could regulate the expression of CCL24 and CCR3 of trophoblasts. We further investigate how trophoblast-derived CCL24 mediates the function of trophoblasts in vitro, and conclude that CCL24/CCR3 promotes the proliferation, viability and invasiveness of trophoblasts. In addition, analysis of the downstream signaling pathways of CCL24/CCR3 show that extracellular signal-regulated kinases (ERK1/2) and phosphoinositide 3-kinase (PI3K) pathways may contribute to the proliferation, viability and invasiveness of trophoblasts by activating intracellular molecules Ki67 and matrix metallopeptidase 9 (MMP9). However, we did not observe any inhibitory effect on trophoblasts when blocking c-Jun N-terminal kinase (JNK) or p38 pathways. In conclusion, our data suggests that trophoblast-derived CCL24 at the maternal-fetal interface promotes trophoblasts cell growth and invasiveness by ERK1/2 and PI3K pathways. Meanwhile, pregnancy-related hormones (P and hCG), as well as DSCs could up-regulate CCL24/CCR3 expression in trophoblasts, which may indirectly influence the biological functions of trophoblasts. Thus, our results provide a possible explanation for the growth and invasion of trophoblasts in human embryo implantation.

[Gene conjugation of molecular adjuvant C3d3 to hCGbeta enhanced the anti-hCGbeta humoral immune response via upregulation of CXCR4 expression on splenic B cells in DNA vaccination].

To investigate mechanism of the anti-hCGbeta humoral immune responsive enhancement by gene conjugation of the molecular adjuvant C3d3 to hCGbeta in DNA immunization, BALB/c mice were inoculated intramuscularly with pCMV4-hCGbeta-C3d3, pCMV4-hCGbeta and pCMV4, respectively. The titers of anti-hCGbeta IgG/IgA antibody in serum were determined by indirect ELISA. The IgG/IgA ASCs levels were evaluated by ELISPOT. The expressions of chemokine receptors on B cells were analyzed by RT-PCR, and CXCR4 expression was analyzed respectively by RT-PCR and FCM. The expression of CXCL12 in spleen was investigated respectively by RT-PCR and ELISA. We found that anti-hCGbeta IgG antibody titer in serum after pCMV4-hCGbeta-C3d3 immunization was significantly higher than that of pCMV4-hCGbeta immunization. But the anti-hCGbeta IgA antibody titers appeared no difference between the two immunized groups. The level of IgG ASCs in spleen of pCMV4-hCGbeta-C3d3 immunization was significantly higher than that of pCMV4-hCGbeta immunization,but the levels of IgA ASCs appeared no difference between the two groups. The expression of CXCR4 on B cell increased after pCMV4-hCGbeta-C3d3 immunization, and significantly higher than that of pCMV4-hCGbeta immunization. The rate of CXCR4+ cell was correlated to the numbers of the ASC (r = 0.966, P < 0.05). CXCL12 expression increased after pCMV4-hCGbeta and pCMV4-hCGbeta-C3d3 immunization, and appeared no difference between the two groups. These results above indicate that gene fusion of the molecular adjuvant C3d3 to hCGbeta can significantly enhance the antigen-specific antibody response, and the level of IgG ASCs in spleen via upregulation of CXCR4 expression on splenic B cells in DNA vaccination.

Molecular adjuvant C3d3 improved the anti-hCGbeta humoral immune response in vaginal inoculation with live recombinant Lactobacillus expressing hCGbeta-C3d3 fusion protein.

To enhance the contraceptive efficiency of human chorionic gonadotrophin (hCG)-beta contraceptive vaccine, we coupled hCG-beta gene with molecular adjuvant C3d3, and cloned into live Lactobacilli (Lb.) to express fusion protein hCGbeta-C3d3. The recombinant Lb. could survive in BALB/c murine vagina for at least 3 weeks. After inoculating BALB/c and C57BL/6 mice via vagina, we found that the antibody titer peaks induced by the Lb.hCGbeta-C3d3 inoculation were higher significantly than the Lb.hCGbeta. T and B cells in spleen and vagina were significantly increased, and anti-hCGbeta IgG and IgA antibody-secreting cells in uterus and vagina were significantly increased compared to the control in different strain mice. Our study shows that the C3d3 can display apparent adjuvant efficiency to induce more powerful humoral response to the hCGbeta antigen in vaginal mucosal immunization.

[Molecular adjuvant C3d up-regulates both B7-1 and B7-2 expression on Raji cells].

To explore modulation of the molecular adjuvant C3d in the hCGbeta -C3d3 fusion protein in costimulatory molecule expression on Raji cells by linking to the surface molecule CD21. Raji cells, B cell line, were incubated with the purified hCGbeta-C3d3, hCGbeta or C3d3 protein for 15 hours respectively in the interference of anti-CD21 monoclonal antibody or not. The culture concentration of each group was 10, 30, or 90 microg/ml respectively. The expression of both B7-1 and B7-2 on Raji cells were analyzed by flow cytometric assay. It was observed that compared to hCGbeta or C3d3 protein, hCGbeta-C3d3 up-regulated both B7-1 and B7-2 expression on the Raji cells, and the effect was in a dose-dependent manner. The up-regulation effect was blocked efficiently by anti-CD21 mAb. The results showed that molecular adjuvant C3d may up-regulate both B7-1 and B7-2 expression on Raji cell via C3d/CD21/CD19 complex, thus enhance the antigen presentation of B cell, and the interaction between B cell and T cell.

Gene conjugation of molecular adjuvant C3d3 to hCGbeta increased the anti-hCGbeta Th2 and humoral immune response in DNA immunization.

Human chorionic gonadotropin (hCG) has been used as an anti-fertility vaccine and as a target for cancer immunotherapy. We have explored the use of three copies of C3d in DNA vaccine as molecular adjuvant to improve the immunogenicity of this hormone in previous work and found that the immune response induced by pcDNA3-hCGbeta-C3d3 has been enhanced 243-fold compared with pcDNA3-hCGbeta following DNA immunization in BALB/c mice. In the present study, a new functionally active DNA vaccine of hCGbeta-C3d3 chimera based on pCMV4 vector has been described. We compared the expression efficiency of pCMV4 and pcDNA3 eukaryotic vectors for hCGbeta and hCGbeta-C3d3 fusion protein and the immune response of mice immunized with pcDNA3-hCGbeta, pCMV4-hCGbeta, pcDNA3-hCGbeta-C3d3 and pCMV4-hCGbeta-C3d3, respectively, at 25, 50 and 100 pmol dose, and further analyzed the levels of Th1 and Th2 cytokines produced by spleen lymphocytes of the immunized mice upon hCG restimulation in vitro. It was found that pCMV4 vector achieved 1.3-1.5-fold higher protein expression and raised 1.1-1.2 (primary) and 1.2-1.3 (booster) logs higher titer of anti-hCGbeta IgG than pcDNA3. Mice vaccinated with 50 pmol of hCGbeta-C3d3-DNAs elicited the highest titer of hCGbeta-specific antibody among the serial doses and the immune response induced by pCMV4-hCGbeta-C3d3 were, respectively, 1.3, 1.3 and 1.2 logs higher than that of pcDNA3-hCGbeta-C3d3 and 2.2, 2.9 and 2.4 logs higher than that of pCMV4-hCGbeta at week 2 following the booster immunization. Moreover, we observed that the production of IL-4 and IL-10 increased in mice vaccinated with hCGbeta-C3d3-DNAs and the ratio of IL-4/IFN-(gamma) showed a Th2 bias of immune response in the mice immunized with hCGbeta-C3d3-DNAs. These findings indicated that gene fusion of C3d3 to hCGbeta, as a means of harnessing the adjuvant potential of the innate immune system, may improve the antigen-specific Th2 humoral immune response of the hCGbeta DNA vaccine and the pCMV4 vector is a more ideal eukaryotic vector for DNA vaccine than pcDNA3.

Mucosal inoculation of Lactobacillus expressing hCGbeta induces an anti-hCGbeta antibody response in mice of different strains.

To show that an anti-human chorionic gonadotrophin-beta (hCGbeta) antibody response can be induced by inoculating Lb. expressing hCGbeta through different mucosal pathways in mice of two strains, female BALB/c and C57BL/6 mice were immunized via vaginal, oral or nasal routes with 10(8), 10(9), and 10(10)Lb.hCGbeta (a recombinant Lactobacillus expressing hCGbeta). The mice were immunized twice with a booster in study week 3. An indirect ELISA was used to determine anti-hCGbeta IgG and IgA antibodies in vaginal lavage and serum, obtained from the 2nd to 8th week after the primary immunization. Flow cytometry was used to analyze the lymphocyte proliferation from these tissues, 1 week after the primary immunization. The hCGbeta antigen-specific antibody-secreting cells of spleen, uterus, and vagina were evaluated by enzyme-linked immunospot assay (ELISpot), 2 weeks after the booster. The analysis showed that 10(9) and 10(10)Lb.hCGbeta inoculations induced similar anti-hCGbeta antibody responses, while the three mucosal pathways induced similar antibody responses. The antiserum obtained after boosters with 10(9) and 10(10)Lb. hCGbeta was able to neutralize more than 100 ng/ml hCG antigen, both in BALB/c and C57BL/6 mice. The highest antibody titer induced by vaginal mucosal immunization was stronger than that obtained via the other mucosal pathways. The B cells in the vagina appeared to proliferate after vaginal immunization (P<0.05). The numbers of anti-hCGbeta IgG and IgA antibody-secreting cells in the uterus and vagina were greater than in the spleen. Therefore, the vaginal mucosal route appears to be a better immunization pathway to induce higher anti-hCGbeta antibody levels in the reproductive tract.

[Enhancement of immunogenicity of hCGbeta protein vaccine and the hCG neutralization capacity of anti-serum by using the molecular adjuvant C3d3].

To enhance the immunogenicity of hCGbeta protein vaccine and the hCG neutralization capacity of anti-serum by using the molecular adjuvant C3d3, the secreted 6his-hCGbeta-C3d3 fusion protein and 6his-hCGbeta were expressed in CHO cells and purified with Ni(2+)-chelating chromatography and Sephadex G-150 column. Then we investigated the potential of three copies of C3d as the molecular adjuvant of hCGbeta protein antigen. The antibody response to hCGbeta-C3d3 conjugates was compared with those resulting from immunization with hCGbeta alone and hCGbeta plus CFA/IFA in BALB/c mice. Our results showed that the antibody titer of hCGbeta-C3d3 was 1995-fold higher than that of hCGbeta alone and the anti-serum was capable effectively neutralizing the bioactivity of hCG. The immunity-enhancing action of C3d3 was 10-fold (primer) and 32-fold (booster) greater than CFA/IFA. These results indicated that C3d3 conjugates might be a better way to overcome the poor immunogenicity of hCGbeta contraceptive vaccine.

Adoptive transfer of paternal antigen-hyporesponsive T cells induces maternal tolerance to the allogeneic fetus in abortion-prone matings.

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/JxDBA/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.

Enhancement of humoral immunity to the hCG beta protein antigen by fusing a molecular adjuvant C3d3.

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 hCG beta protein antigen. The antibody response to the hCG beta-C3d3 fusion protein immunization was compared with those resulting from immunization with the hCG beta alone and the hCG beta plus CFA/IFA either in BALB/c mice or in C(57)BL/6J mice. Our results showed that the fusion of C3d3 to hCG beta protein antigen resulted in a significant elevation of the serum anti-hCG beta 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 hCG beta, as a means of harnessing the adjuvant potential of the innate immune system, may improve immunogenicity of the hCG beta contraceptive vaccine, which is useful to produce a cost-effective vaccine and for the less-responsive population.

Inoculation of Lactobacillus expressing hCG beta in the vagina induces an anti-hCG beta antibody response in murine vaginal mucosa.

OBJECTIVES: To test the possibility of vaccination with lactobacillus expressing hCG beta antigen administered by vaginal mucosal immunization. METHODS: A plasmid pIlac-hCG beta was constructed and then transfected into Lactobacillus casei CECT5276, which stably expressed hCG beta protein. RIA was used to detect hCG beta 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-hCG beta 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-hCG beta, and the highest transfection efficiency of both plasmids was at pulse voltages of 1200 V and 1500 V. About 78.5% of the hCG beta protein was excreted into the culture supernatant. Excretion of hCG beta 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 beta protein in the vaginal lavage fluid of these BALB/c mice was positive on the third day after vaginal inoculation. Anti-hCG beta 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 hCG beta through the vagina underwent a proliferative reaction to hCG antigen restimulation in vitro. Interferon gamma (IFN-gamma) and interleukin (IL)-4 were secreted at higher levels after vaginal mucosal immunization of L. casei expressing hCG beta than after vaginal mucosal immunization of L. casei alone. CONCLUSIONS: Vaginal immunization of lactobacillus expressing hCG beta induced an anti-hCG beta 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.

[C3d molecular adjuvant increases the immunity of human chorionic gonadotropin beta DNA contraceptive vaccination and changes its immune response from Th1 to Th2].

OBJECTIVE: To testify the effect of C3d molecular adjuvant on the immunogenicity of human chorionic gonadotropin beta (hCG beta) DNA vaccination as well as the mode of immune response. METHODS: BALB/c mice aged 6 weeks were immunized intramuscularly two times at an interval of 3 weeks with by the plasmid pcDNA3 (A1-3 groups), pcDNA3-hCG beta (B-3 groups), pcDNA3-hCG beta-C3d3 (C1-3 groups), or pCMV4-hCG beta-C3d3 (D1-3 groups), at dosage of 5 pmol, 10 pmol, and 20 pmol, respectively. Three weeks after the second vaccination the animals were killed, specimens of their peripheral blood were extracted to determine the anti-hCG beta antibody titer by indirect ELISA. Their spleen cells were harvested and stimulated in vitro by hCG antigen for 24 hours. The Th1/Th2 cytokines in the culture supernatant were determined by ELISA. RESULTS: At the dosage of 20 pmol, C3d molecular adjuvant significantly enhanced the anti-hCG beta antibody titer. The utmost anti-hCG beta antibody titer of C3 group was 1:450, 9 times higher than that of B3 group, and the utmost anti-hCG beta antibody titer of D3 group was 1:12 150, 243 times higher than that of B3 group. Stimulated in vitro by 5,000 IU hCG beta antigen, the splenic cells of the C3 and D3 immunization group produced significantly lower IL-2, INF-gamma and TNF-alpha than those of the B3 immunization group (P < 0.01 or P < 0.05). The IL-4 level of the C3 group was higher than that of the B3 group while the IL-10 level of the D3 group was significantly higher than that of the B3 group (P < 0.01). CONCLUSIONS: The C3d molecular adjuvant increases significantly the hCG beta immunogenicity of hCG beta DNA vaccination; meanwhile decreases the secretion of Th1 cytokines (IL-2, INF-gamma, and TNF-alpha), and increases the expression of Th2 cytokines (IL-4 and IL-10) in response to hCG antigen. So C3d changes the anti-hCG immune response from Th1 type to Th2 type.

Gene fusion of molecular adjuvant C3d to hCGbeta enhances the anti-hCGbeta antibody response in DNA immunization.

OBJECTIVE: To express the hCGbeta-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-hCGbeta-C3d3 which contains three copies of murine C3d cDNA and the hCGbeta gene by cloning the chimerical hCGbeta-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 hCGbeta 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-hCGbeta and pcDNA3-hCGbeta-C3d3, and ELISA was used to assess anti-hCGbeta IgG antibody in serum. RESULTS: In 72 h after COS-7 cells were transfected with the plasmid pcDNA3-hCGbeta-C3d3, 1.0x10(5) cells could secrete 152 ng of the recombinant protein (calculated by hCGbeta contained). The transfected CHO cells, which were then screened by G418, could continuously secrete the fusion protein at 660 ng/10(6) cells/48 h. The hCGbeta-C3d3 protein was purified by anti-hCGbeta immunoaffinity chromatography. Raji cell immunohistochemical assay demonstrated that both the hCGbeta and C3d3 were successfully fused. After DNA immunization intramuscularly, the anti-hCGbeta IgG antibody titer in the pcDNA3-hCGbeta-C3d3 immunized group was 243-fold higher than that of the pcDNA3-hCGbeta immunized group. CONCLUSION: We have expressed the hCGbeta-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 hCGbeta antigen in DNA immunization.