ABSTRACT
Objective To compare the cellular immune responses induced by two different vectors,eukaryotic plasmid pcDN3.1(+) and attenuated typhia live vaccine Ty21a,-associated mimic epitopes of HCV hypervariable region 1,so to find a better way for vaccine immunization.Methods The DNA sequence based on the peptides' sequence of HCV hypervariable region 1 related consensus polymimotopes was synthesize and then inserted into plasmid pcDN3.1(+) to construct recombinant plasmid pcDN3.1(+)-SP,which then was transfected into live attenuated typhia vaccine Ty21a to construct Ty21a-SP.The mice were immunized orally with Ty21a-SP and intramuscularly with pcDN3.1(+)-SP,respectively,and then sacrificed by exsanguination.The splenocytes were separated and restimulated with pooled synthesized peptides,and then collected.Flow cytometry was employed to identify CD8+IFN-?+ T cells,and non-radioactive MTS method was adopted to test T cell proliferation,and non-radioactive LDH method was used to test cytotoxic T cytolytic reaction(CTL).Results Compared with control groups,the proliferation of splenocytes was apparently enhanced,the proportion of CD8+IFN-?+ cells obviously increased,and CTL responses also significantly increased after the spenocytes of mice immunized by pcDN3.1-SP and Ty21a-SP were restimulated with synthesized peptides.And the responses mentioned above in the mice immunized by Ty21a-SP were stronger than those mice immunized by pcDN3.1-SP.Conclusion Using attenuated typhia live vaccine Ty21a as DNA vector is an effective way to induce cellular immune responses.
ABSTRACT
Objective To clone CD34 extracellular region encoding cDNA and to construct its eukaryon expression vector to explore the feasibility of its monoclonal antibody preparation by gene immunization. Methods Total RNA was extracted from KG-1a cell lines. CD34 extracellular region encoding genes were amplified by RT-PCR method and then confirmed by enzymatic lysis and DNA sequencing, and its eukaryon expression vector was constructed as pcDNA3.1-CD34. Twelve BABL/c mice aged 4-6 weeks were selected and randomly divided into 3 groups. Before immunization, 50?l 25% saccharin solution was injected into mouse musculus quadriceps femoris. Fifteen minutes later, blank control PBS, PBS diluted empty vector pcDNA3.1(50?g), or PBS diluted pcDNA3.1-CD34 was injected into the same site of the above three groups, respectively. Immunization was taken every two weeks for a total of three times. The antibody was detected regularly by FACS using tail blood from immunized mice. Results The results demonstrated that the length of CD34 cDNA was 886bp which was identical to the theoretical value and its sequence was confirmed by DNA sequencing which was identical to the registered sequence. The accuracy of CD34 expression vector recombination was confirmed by restriction enzymatic lysis. Hind III and EcoRI restriction enzymatic lysis sites were introduced into 5 and 3 terminals of amplified cDNA sequence, respectively. Terminate code TGA was also introduced into CD34 extracellular encoding cDNA. The expression vector possessing target genes was named as pcDNA3.1-CD34. FACS detection indicated that only 1/4(25%) immunized mice had a lower titer CD34 antibody in their tail vein serum 2-6 weeks after immunization, and the others did not show no antibody production. Conclusion The eukaryon expression vector pcDNA3.1-CD34, which express CD34 extracellular region, has been constructed. The feasibility of CD34 McAb preparation can primarily be confirmed by gene immunization.
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Objective: To study the anti-tumor efficacy induced by antibodized tumor epitope PDTRP gene immunization. Methods: Three copies of tumor associate gene PDTRP from MUCI tandem repeats were designed and mimicked the conformation of MUCI by Insight Ⅱ . The ?lneo-PDTRP plasmid was further constructed, in which the PDTRP target gene was inserted into CDR3 of the ?1 -neo vector. The specific humoral and cellular immune responses towards to PDTRP were detected after intraspleen immunized Balb/c mice with "ylneo-PDTRP. And the immune protection assay was also done to observe whether the mice immunized with ?lneo-PDTRP could prolong the survival after tumor challenge. Results: The conformation of three copies of PDTRP mimicked the conformation of MUCI tandem repeats. The expression of ?lneo-PDTRP could be detected after in vitro transfect. The specific antibody against PDTRP epitope could be induced and increase to a higher titer after intraspleen injection with a ?lneo-PDTRP plasmid. And the specific proliferation and cytotox-ic function of lymphocyte were also increased. There is a significant survival from mice immunized with ?lneo-PDTRP a-gainst the 4T1-PDTRP tumor challenge. Conclusions: Gene immunization with ?lneo-PDTRP could elicit both humoral and cellular tumor specific immune response and had the protective effect.
ABSTRACT
Objective: To investigate whether the plasmid ?1neo-hgp100 could be expressed and presented in vitro and could protect the immunized mice from B16F10 challenge in vivo. Methods: ?1neo-hgp100 plasmid was constructed in which the DNA sequence encoding hgp100 CTL epitope inserted into CDR3 of ?1-neo vector. The expression of anti-bodized antigen and IFN-? in supernatant was measured by ELISA respectively after transfection J558L with ?1neo-hgp100 and further co-culture of J588L transfacted with ?1 neo-hgp100 and pmel TCR transgenic T cell. After introspleenic inoculation of ?1neo-hgp100, the protective efficacy of the gene vaccine was observed by means of measuring the tumor area every two days. Results: ?1neo-hgp100 could be expressed and presented in vitro, the immunogenecity of CTL epitope of hgp100 was strong enough and could activate gp100 specific T cell, the mice immunized with the gene vaccine could resist the tumor challenging in vivo. The mean survival time was prolonged to 36 days, compared to control group (P