| Object The malignant tumor is a serious disease that greatly hurts human health and life. Because of lacking efficient and sensitive therapeutic means, recurring and transferring of tumor can not be inhibited. It is overwhelming urgent to discover improved therapeutic methods. The biological therapy with low toxicity and side-effect was remarkable. The immune therapy is one important method of biological therapies. Up to now, there was great progress in anti-idiotypic vaccine > antigen vaccine and DNA vaccine, but there are some defects in it, not reaching the expectant effect. The bacteriophage display attracts dramatically attention of people, and it can express inserted gene or a DNA fragment to form fusion capsid protein with primary dimension structure and biological activity. The epidermal growth factor receptor (EGFR) protein is a tumor-associated antigen (TAA) recognized. The tumors originated from the epithelial tissue have EGFR over-expression to a certain extent. The monoclonal antibody has been used for treatment of EGFR-positive expression tumors and entered clinical trial. Therefore, the recombinant bacteriophage T7 vaccine was constructed targeting human EGFR. The immune reactivity of anti-tumor was studied in our experiment both in vitro and vivo, for providing a basis of study on anti-tumor vaccine.Materials and Methods Checking GenBank databases, according to theDNA sequence of EGFR extracellular region, a pair of primers were designed by use of primer-premers 5.0 version software. About 1900bp DNA fragment was cloned from A549 lung cancer cells with expression high level EGFR. Another pair of primers including EcoRI or Hindlll restriction endonuclease site at respectively 5"-ends were designed, and about 1800bp fragment was subcloned. The subcloned fragment was ligated into pMD18-T cloning vector. The recombinant pMD18-T vector was transformed into compenent cells. The plasmid DNA was extracted, and the target DNA fragment was sequenced by Genecore Biological Company in Shanghai. Based on the limited length of inserted DNA fragment and functional difference in domains of EGFR extracellular region; the peptide domain with better effect than that of the antigen mimic was determined by using proteins analysis software, Three pairs of primer including restriction endonuclease sites of both EcoRI or Hindlll were designed, and the three DNA fragments were subcloned using identified plasmid as the template. Three subcloned fragments were ligated into bacteriophage T7 vecter arms, and the recombinant bacteriophage T7 was packaged in vitro. The recombinant bacteriophage T7 was transfected into a host strain, amplified and purified. A large amount of recombinant bacteriophages T7 were harvested. The recombinant bacteriophage T7 were detected with PCR technique ^ SDS-PAGE and Western-blotting. The immune reactivity was demonstrated after injecting the recombinant bacteriophage T7 into the animal. The titer of specific antibody was detected with Dot-blotting. The serum from immunized mice could recognize the A431 squamous cancer cells with high expression EGFR by flow cytometry. While the isolated spleen cells from immunized mice acting as effector cell, and A431 cells as the target cell, the specific cytotoxicity was detected with MTT.Results A 1900bp fragment from A549 cell was cloned by RT-PCR, in addition, about 1800bp fragment of EGFR extrecellular region was subcloned, and subcloned fragment was ligated into pMD18-T cloning vector. The recombinant pMD18-T vector transformed into the compenent DH5a, and some bacteria colonies were found on the agar- LB culture plate containing amphicillin. A single white colony was extracted and proliferated in LB, then the plasmid DNA was extracted,and the DNA fragment was sequenced. The result showed that there were no any inserted and deleted mutation. Three DNA fragments, 90bp in 1075-1165 site, 132bp in 546-678 site and 638bp in 625-1263 site in the EGFR extracellular regions, were subcloned and were ligated into T7 vector arms respectively. Two high-copy number recombinant bacteriophages T7 and one low-copy number recombinant bacteriophage T7 were constructed in the experimental group, named as T7415-lb-90 > T7415-lb-132 and T710-3b-638 respectively. In the control group, a recombinant bacteriophages T7 was constructed (insertion of 45bp fragment expressing S.Tag peptides from pancreatic ribonuclease A). The recombinant bacteriophages T7 were identified. It is determined that the target DNA was cloned with PCR by use of the UP/DOWN primers (in the kit). The SDS-PAGE results showed that the molecular weight of the fused proteins of high-copy and low-copy number recombinant bacteriophages T7 were about 40kD and 60kD respectively. The Western-blotting with polyclonal antibody showed that the experimental groups were all positive, and the control recombinant bacteriophage T7 was negative. In the experimental groups. 1:2000 titer exhibited in the immunized mice were detected by Dot-blotting, the strongest signal in the T7415-lb-132 group and 1:1000 titer exhibited positive signal in the T7415-lb-90 and T710-3b-638 group. The positive A431 cell percentages in the immunized serum were 37.6%, 47.6% and 35.1% respectively detected with flow cytometry. In addition, the ratio of effector cells versus target cells was 10:1, the cytotoxicity was distinct. The CTL activity percentages for cytotoxic effects were 26.50%, 29.47% and 0.2% respectively. The ODE+Tby ELISA Reader possessed significant difference in the experimental groups in comparison with the control groups (p<0.01). It had no significant difference in ODe and ODT between the experimental and the control groups.(p>0.05)Conclusions: ?The target DNA fragment of EGFR extracellular region was successfully cloned from A549 cells. ?A recombinant pMD18-T vector was successfully constructed, and the sequence of target DNA fragment was correct. ?The target DNA fragment's directly inserted into bacteriophage T7 expression vector arms, and the recombinant bacteriophage T7 in experimental group with primarydimension structure and function activity was constructed, and could be recognized efficiently by the polyclonal anti-human EGFR protein as the ligand. ?The immunized mice could produce both humoral immune response and cellular immune response. The immune serum could not only recognize the human EGFR protein from human A431 squamous cancer cells, but also the EGRF protein in dimension structure on the surface of A431 cells. (§)CTL response could be induced in the immunized mice in two high-copy number recombinant bacteriophages T7, lots of spleen cells from immunized mice gathered surrounding A431 cells were found under inverted microscope. The cytotoxic effect was higher in the T7415-lb-132 recombinant bacteriophage T7. The subcloned 132bp fragment at the combined domain of ligand may be the active domain with motifs in EGFR extracellular region. Both the humoral immune response and the cellular immune response was the strongest in immunized mice with T7415-lb-132 recombinant bacteriophage T7. ?It was relatively safe and had no marked toxicity and side effect in the immunized mice. ?The bacteriophage vaccine may be an efficient and practical anti-tumor vaccine. |
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