Structure Of Recombinant Adenovirus Encoding Her-2/neu Gene Of Extracellular And Transmembrane Domains Protein And Their Eliciting Immunological Response

Malignant tumor, a common disease to threaten public health, is the 1 st/2nd cause of human death that is about seven million worldwide every year. The incidence of malignant tumor goes up gradually in recent years and patients tend to become younger and younger. World Health Organization foresees that it will be "No.l killer to human " in 21st century. Its therapy has always been the hot spot that people show solicitude for. Immunotherapy, besides traditional surgical therapy, radiotherapy and chemotherapy, plays more and more important role in the treatment of malignant tumor at the present. Anti-tumor vaccine has presented tempting prospect for people, specially the carrier vaccine of genetic engineering raised in recent years. Her2/neu, one of the tumor-associated antigens, over-expressed in many epithelial tumors, such as breast cancer, ovary cancer, gastric cancer and lung cancer, has show potential as targeting antigen of vaccine. Type 5 adenovirus, which is safe and high-efficacy, takes dominant position in vector for gene delivery. In this study we developed a Her2/neu vaccine and then investigated its immunogenicity by rAdenovirus vector expressing Her2/neu proteins of extracellular and transmembrane domains. Meanwhile, we also have estimated the feasibility that rAdHer2 was introduced to dendritic cell-based immunotherapy of anti-tumor.Genes of Her2/neu extracellular first-receptor domain(Her2-ECDs), full-length extracellular domain(Her2-ECD), extracellular and transmembrane domain(Her2-TM) proteins were amplified from total RNA resulted from SK-BR-3 cells over-expressing Her2/neu by RT-PCR, and then cloned into the PMD-18T vector after adding A. The right sequences were identified by sequencing with Ml3 primers and a lowest frequency (1.2%) of4occurrence of Her2/neu gene polymorphism was detected that both of the first and the second amino acids of transmembrane domain protein were Valines. During the second cloning, objective genes cut off from PMD-18 vectors were inserted into shuttle pAdTrack-CMV plasmids and then rAdHer2-ECDs, rAdHer2-ECD and rAdHer2-TM plasmids were obtained from homologous recombination in E coli. BJ5183, into which the Adeasy?plasmid of adenoviral framework and the recombined pAdTrack-CMV plasmid were co-transfected. rAdHer2-ECDs , rAdHer2-ECD and rAdHer2-TM were isolated by screening after linearized rAdHer2-ECDs, rAdHer2-ECD and rAdHer2-TM plasmids were transfected into 293 package cell line with polyfect. Virus with 5.4VP/ml, 2.95 10l2VP/ml and 8.40 1012VP/ml titre, respectively, measured by physical method, were generated after large scale virus amplification in 293 cell and purified by Cesium Chloride Gradient. The CPE didn't achieve in Hela cell undergone 3 passages, after infected by purified recombined adenovirus. The express of proteins was probed by Western Blot Check in infected 293 cell, and the amounts of Her2/neu elevated with the increase of MO. These results indicated that generated recombined adenovirus with high purification and titre can transfer package cell line efficiently in vitro.To estimat the transduction efficiencies of rAdHer2-ECDs , rAdHer2-ECD and rAdHer2-TM in vivo and their ability of eliciting immune response, The Balb/c mice were infected by recombined adenovirus via intravenous injection(IV), intrauscular injection(IM), intraperitoneal injection(IP) and gastric gavage(gg). The results showed that there is no significant difference in Her2 antibody titres among via the same administration route. The titre level of Her2 antibody induced via intravenous injection was the highest in same recombined adenovirus among the different administration routes, following with intramuscular injection and intraperitoneal injection route in turn, but no difference was detected between them, and that of anyi-Her2 antibody via gastric gavage was the lowest. The CTL responses induced by recombined adenovirus were same as the administration of rAdHer2-ECDs , rAdHer2-ECD or rAdHer2-TM via different routes. rAdHer2-TM can induce the strongest CTL response via...