Research On Immune Protective Mechanism Of Gene Vaccines With Co-expressing Bcr-abl Fusion Gene Fragment And Mouse IL-7

The bcr-abl fusion gene is the characteristic gene of chronic myeloid leukemia (CML). We had cloned bcr-abl fusion gene fragment about 472bp into plasmid pcDNA3.1 and constructed pcDNAbcr-abl. The BALB/c mice immunized with pcDNAbcr-abl by muscle injection could elicit to produce specific antibodies to fusion protein of p210~bcr-abl. We decided to study the influences of mIL-7 on the immune responses by vaccine of bcr-abl fusion gene fragment in mouse. The research was divided into three parts.1. Construct and express co-expressing vector of bcr-abl fusion gene fragment and mouse IL-7 geneWe have cloned bcr-abl fusion gene fragment about 472bp and mouse IL-7 gene into the vector pVITR02-mcs. The fragment of bcr-abl fusion gene and mouse IL-7 gene were promoted with different promoter respectively. The recombinant plasmid pVbcr-abl/mIL7 was transfected into CHO cells with liposome. It was confirmed that fragment of bcr-abl fusion protein was expressed in cytoplasm of CHO cells transiently by IFA. And we detected mRNA expression of mIL-7 gene in CHO cells by RT-PCR. It provided a new experimental tool to study bcr-abl gene vaccine.2. Influences of two bcr-abl gene vaccines on mice inoculated SP2/0/bcr-abl tumor cellsBALB/c mice were immunized with pVbcr-abl, pVbcr-abl/mIL7 plasmids respectively, then inoculated subcutaneous SP2/0/bcr-abl cells which express the fragment of bcr-abl fusion gene into the abdomen of BALB/c mice. We found that there were dramatic differences on the appeared time of tumor growth, the appeared time oftumor ulcer and tumor volume, survival time between two immunized groups and the control groups. The mice immunized with pVbcr-abl/mIL7 lived longer than that with pVbcr-abl. In HE tissue slice, the tissue of subcutaneous tumor was looser in immunized groups than in control groups and there were many infiltrated lymphocytes. Moreover, there were migrating tumor cells in the livers of control groups. The results suggested that the immunized group generated specific immune protection that could inhibit the growth of SP2/0/bcr-abl tumor cell.3. Initial research on immune protective mechanism of two bcr-abl fusion gene vaccinesThe BALB/c mice immunized with pVbcr-abl/mIL7 ^ pVbcr-abl by muscle injection respectively could elicit to produce higher specific antibodies to the protein of p210 cr"a . The specific antibody level of former group was higher than the latter group but there was no statistic difference. The spleen cells from the immunized mice had more effective CTL activity than the control group. The cytotoxic activity of CTLs in spleen induced by pVbcr-abl/mIL7 immunized mice exceeded that of pVbcr-abl immunized mice, and there was no statistic difference as well. We inspected the component of T cells in spleen by FACS and found that the ratio of CD4+/CD8+ was higher in immunized groups (2.09±0.24 and 1.54+0.29 respectively), exceeding the control group (1.18 + 0.30). CD4+ T cells were dominant in the spleen cells of immunized mice. CD3+ T cells infiltrated into inoculated tumor tissues of the mice immunized with pVbcr-abl/mIL7> pVbcr-abl. The number of CD3+ T cells were 62.4 + 7.44, 39.1+2.38, respectively. It was apparent that the number of CD3+ T cells in pVbcr-abl/mIL7 immunized group exceeded that in pVbcr-abl immunized group. And the ratio of CD4+/CD8+ was 0.92 and 0.89 respectively. It suggested that infiltrated CD8+T cells were dominant in tumor tissue. We guessed that mIL-7 could influence growth and differentiation of T cells, promote some T cells migrating to the tumor tissue and up-regulate the specific cellular immune response.