Construction Of The Tumor-Specific Imagining System And Trans-Kingdom Thya Homologous Recombination Vector In Lactococcus Lactis

BackgroundTumor markers have been applied in clinic for years, which played animportant role in early diagnosis, follow-up and therapy evaluation of cancers.Because of poor specificity of traditional tumor marker, some benign non neoplasticdiseases often show a false positive. Moreover, most of the tumor markers onlyconfined to a particular tissue type or several types of cancers, but not widelyapplied to different types of tumor tissue. In order to improve the accuracy andcomprehensiveness of inspection, we always make a variety of serum tumor markersinto a combination, then detect different groups of markers depending on thedifferent tissue types of tumors. As a clinical screening tool, the method above is alarge economic investment, which doesn’t match the cost–effective principle.With excellent targeting, PEG-3(progression elevated gene-3), which is apromoter of a wide range of tumors, can specifically recognize a variety of malignant cells without affecting normal cells. By reason of high level gene transferand expression, most of the researches on PEG-3mediated exogenous geneexpression used viral vectors. However, they can cause a strong inflammatoryimmune response in the host and there production costs are high. In addition, themost important is that virus vectors have potential danger.Some researches show that it can be safe and effective to mediate exogenousgenes into the mammalian cells by Bactofect. In other word, combined the Yersiniapseudotuberculosis invasin gene (inv) with the Mononucleosis lester bacteriahemolysin gene (hly) can make the bacteria had ability to enter the mammaliannon-phagocytes and express the prupose gene, although this bacteria can not attactcell inherently.This method has been confirmed on E.coli and S.aureus. Lactococcuslactis (L.lactis) is generally regarded as safe (GRAS) bacteria. Hence, it is the bestchoice for genetic engineering to deliver interesting recombinant peptide or protein.Vectors of L.lactis almost have antibiotic resistance as selective marker,erythromycin and chloramphenicol resistance for instance. Therefore, it has a higherbiological safety by using no antibiotic resistance selective marker——thyAdeficient bacteria, due to thyA deficient in the L.lactis could not survive in theenvironment. It can avoid the qualms about horizontal gene transfer of exogenousgenes.If we combine L.lactis thyAˉwith tumor-specific imagining system, thetrans-kingdom recombinant bacteria can invade into mammalian cells, express andsecrete green fluorescent protein. This method enables to visualize and quantify thetumor cells. The implications of this finding would not only aid tumor detection, butalso extend to pre-operative planning, intra-operative management and therapeuticmonitoring. Additionally, by combining this unique delivery methodology withtherapeutic agent, it would be possible to realize the targeted cancer therapy.Objective:1、Construct the Tumor-specific imagining system with the lactococcus lactishuttle vector, which open a new field for the study of Tumor-specific oralvaccine. 2、According to the principles of double-cross homologous recombination, wecontrast a homologous recombinant plasmid containing the inv gene, hly gene,upstream and downstream of thyA gene. We preliminary confirmed theinvasive ability of recombinant L.lactis by infecting experiment which lays asolid foundation for constructing thyA deficient recombinant L.lactis in nextstage.ContentThe study was divided into two parts:Part I：Construction and identification of the tumor-specific imagining systemMethods:The PEG-3promoter were constructed by means of the PCR base geneassembly, and conformed by emzyme digestion and DNA sequencing. Thenconstructed the recombinant pTR-peg3-egfp (shuttle plasmid) carrying the PEG-3promoter, egfp (enhanced green fluorescent protein) and poly A tail. Finally, theability of Tumor-specific imaging was validated by means of the transfection invitro.Results:The460bp purpose fragment obtained by PCR gene splicing is conformedcompletely. The tumor specific plasmid pTR-peg3-egfp was constructedsuccessfully, which is got a preliminary validation on cellular level.Conclusion:Vector with PEG-3promoter could specifically recognize tumor cell. Thebrand-new L.lactis expression system can lay a solid foundation for both cancerimaging and tumor-specific gene therapy.PartⅡ construction and preliminary confirmation of the trans-kingdom thyAhomologous recombination vectorMethods: inv gene was cloned into temperature-sensitive plasmid pVE6007by thein-fusion technology. Then, upstream of thyA gene, hly gene, and downstream ofthyA gene were also cloned into the plasmid pVE–inv in sequence to constructhomologous recombination plasmid pVE-inv-hly. The individual bacterial colonyfirstly screen by PCR, the positive clones were subsequently identified by restrictionenzyme digestion. After electroporation of L.lactis, we then made the liver cancercell HepG2and lung cancer cells A549infected by the pVE-inv-hly plasmid inL.lactis and the pVE6007plasmid (excluding inv, hly) in L.lactis respectively, whichhad preliminary confirmed the invasive ability of trans-kingdom recombinantL.lactis.Results:The inv sequence is completely conformed by sequencing. The vectorpve-inv-hly containing the inv gene, hly gene, upstream and downstream of thyAgene was constructed successfully. The infection experiment showed that theL.lactis with pve-inv-hly vector could invade mammalian cells, while L.lactis withpVE6007can not.Conclusion:The homologous recombination vector pVE-inv-hly was construstedsuccessfully. It lays a solid foundation for constructing trans-kingdom thyA deficientrecombinant L.lactis in the future.