| CEA is probably the most extensively characterized human tumor associated antigen. It was discovered in colorectal and pancreatic carcinomas in 1965 and it' s full cDNA was cloned in 1987. It is a kind of Glycoprotein and it' s molecular weight is 180KD. It is normally expressed in human embryo and is important to the growth of embryo and the development of tissue. CEA is generally reduced with the growth of embryo and vanished in the adult. But it is extensively expressed on the majority of colorectal , gastric, and pancreatic carcinomas, and non-small cell lung cancers in the adult. So, CEA can be used to the target of the tumors' detection and treatment in the adult. Although the above tumor cells express CEA, it can't induce the immune response of the body, So, tumor cells can avoid the immunesurveillance of the body and survive.In the first of 1990s, a new kind of vaccine-DNA vaccine gives a new way to solve the immunogenicity of CEA. It is defined as plasmids encoding antigen protein which can elicit immune responses against the antigen through direct injecting into body and taken into by cells, Then, it stimulates the body generate specific immune response through different routes. In 1994, Conry firstly constructed CEA DNA vaccine, studied its immunological effects and made great achievements. In order to enhance DNA vaccine efficacy, researchers are inclined to employ effective and safe vectors, co-delivery cytokines recombinant or adhesion molecules, ISS, and so on. In this study, we have constructed a new CEA DNA vaccine and a plasmid of IL-2 by using Genetic-engineering technology, and designed the immunostimulatory DNA sequence (ISS). We hope enhance the quantity of CEA and the anti~CEA by using the various vector, cytokine and ISS. Materials and Methods:1. Construction, preparation and quantity of CEA DNA vaccine: Plasmid pGEM4-CEA containing full-length of CEA cDNA was cleaved by EcoRI, 2.4kb fragment was recovered, and ligated with EcoRI-cleaved plasmid pCI-neo. The ligation product was then transformed into E. coli DH5 a . Therecombinant was screened by PCR, cleaved by BamHI, Bglll to determine the director, and then was named pCI-CEA. After growing in large scale, plasmids were extracted and purified and quantified.2. The construction of pCI-IL-2: Primers were designed by the software Oligo, Xhol and Sail were added to the primers, the cDNA of IL-2 was obtained by PCR. The cDNA of IL-2 was ligated with pCI-neo cleaved by Xhol and Sail. The recombinant was evaluated by PCR.3. DNA immunizition: Bupivacaine in concentration of 0. 75% was injected in the Tibialis anterior muscle of mice, Three days later, CEA DNA vaccines were injected, each side 50 jag, with different adjuvant (ISS or pIL~2). There are 5 mice in each group. The control group was injected with PBS buffer in a dose of 50 ju 1. On the tenth day, the twenty-fourth day, each group was boosted again.4. Production of anti-CEA and CEA induced by CEA DNA vaccine: On 1 2 4 6 weeks after the last immunization, sera of the immunized mice were collected, ELISA was conducted to detect the anti-CEA and CEA. Experimental data were disposed by software SPSS10. 0.Results:1. pCI-CEA cleaved by EcoRI, the 2.4kb and 5.4kb fragment can be obtained ;Cleaved by BamHI, the 3.0kb and 4.8kbfragment obtained; and cleaved by Bglll, the 0.4kb and 7. 4kb fragment obtained.2. pCI-IL-2 cleaved by Xhol, the 6.4kb fragment can be btained; Cleaved by Xhol and Sail, the 0.8kb and 5.4kb fragment obtained; and cleaved by Banll, the 0. 9kbs 3. 2kb and 1.3kb fragment obtained .3. Compare pCI-CEA vaccine with pcDNA3-CEA vaccine, the former is stronger than the latter in inducing the expression of CEA. pCI-IL-2 and ISS can' t obviously enhance the expression of CEA.4. Production of anti-CEA: The group of immunized with pCI-CEA is stronger than the group of immunized with pcDNA3-CEA. Both ISS and pIL-2 can enhance the quantity of anti-CEA.The findings of this experiment indicate: vector, cytokine and ISS can enhance the ef...
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