| Bursin, crucial to the production of antibody in chicken, is a tripeptide isolated from the bursa of Fabricius. Bursin can stimulate pre-B cells to differentiate and proliferate, and act on the class switching from IgM to IgG. Up to the present time, how bursin signal be introduced into cell is not known. In order to explore the further influence of bursin on production of antibody and their mechanisms, we examined the effect of bursin on the antibody production and the proliferation of E8 hybridoma cells secreting monoclonal antibody (McAb) against human IgG, and the potential bursin signal transduction pathway in this paper. All studies were listed as below:1. Effect of bursin on the proliferation and the antibody production rate of E8 cells. In the present study, it was not observed the effect of bursin on the proliferation of E8 cells analyzed by MTT assay. While it was found that bursin could significantly promote the McAb secretion of E8 cells in dose and time dependent manner by quantitative enzyme-linked immunosorbent assay(Q-ELISA). The production rate of McAb in E8 cells treated with 50μg/mL bursin has reached the highest and was up to 3.7 times of that of the control cells among 2 to 3 hours. These results indicated that bursin could promote the McAb production. In addition, this promotion function was not correlated to the cell proliferation.2. Effect of bursin on the expression of IgG1 mRNA in E8 cells. IgG1 was the class of McAb secreted by E8 cells identified by ELISA. Then the γ1 CH3 fragment amplified by RT-PCR was cloned and sequenced. The nucleotide and amino acid sequence homology of cloned gamma1 CH3 fragment and the original γ1 CH3 in Genbank are 99.1% and 98.2% respectively. The γ1 mRNA contents in E8 cells untreated and treated with bursin were measured by quantitative RT-PCR using beta-actin as aninternal standard. Bursin could up regulate the expression of γ1 mRNA at time and dose dependent manner, the expression level of γ1 mRNA in E8 cells is up to the highest 60 minutes after 50μg/mL bursin-treated, which is near three times of that of the control. These results clearly showed that bursin could up-modulate the expression of McAb mRNA in transcription level.3. Influence of signaling inhibitors on the production of McAb and the expression of γ1 mRNA in E8 cells challenged by bursin. E8 cells were pre-treated by the different specific signaling inhibitors for 30 minute, then challenged by 50μg/mL bursin for 2 hours and 3 hours respectively for antibody levels tested by quantitative ELISA, and 60 minutes for mRNA expression levels measured by quantitative RT-PCR. It was 4 - Cyano-3-M-thylisoquinoline, Genistein and PD98059, the specific inhibitors of protein kinase A (PKA), tyrosine protein kinase (TPK) and mitogen-activated protein kinase kinase (MAPKK) respectively, that interdicted almost completely the effects of bursin on up-regulation of the expression and production of McAb. The inhibit rate of 4 - Cyano-3-M-thylisoquinoline, Genistein and PD98059 on the production rate of McAb in E8 cells were 74.55%, 91.13% and 80.69% respectively. The results indicated that the promotion function of bursin on antibody production and expression was realized through the activation of PKA, TPK and MAPKK and their corresponding signal transcription factors.4. Effect of bursin on PKA activity in E8 cells. PKA in E8 cells was activated by bursin in time-dose dependent manner. PKA activity levels in E8 cells were increased to the peak of 0.821±0.053 pmol/min/μg protein after two minutes stimulated with 50μg/mL bursin, then declined to nearly original level of 0.305 + 0.053 pmol/min/μg protein ten minutes later. Both Genistein and PD98059 could not inhibit PKA activation induced by bursin. According to the results, we found that PKA composed of a signal pathway either alone, or together with TPK and MAPKK as an upstream kinase.5. Effect of bursin on Membranous-TPK (m-TPK) activity in E8 cells. TPKs represent a diverse group of protei...
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