Construction And Gene Immunization Of Recombinant DNA Vaccine PEGFP-M-S Containing Seoul Virus M Segment And Hantaan Virus Partial S Segment

Objective Hemorrhagic fever with renal syndrome (HFRS) prevailed in China was caused mainly by Hantaan virus and Seoul virus that belonging to Hantavirus genus. Most epidemic areas of HFRS were mixed with both viruses but one predominant. So only single serotype of Hantavirus vaccine can't prevent cross infections besides other hantavirus completely. It's in great need to study and develop double valence or multivalence hantavirus vaccine. 1 , In part I of this study, a eukaryotic plasmid vector pEGFP containing Kanamycin resistance gene was used to construct a recombinant DNA vaccine pEGFP-M-S, which containing both Seoul virus Z37 strain whole M segment and Hantaan virus international standard 76-118 strain partial S segment encoding nucleocapsid protein N-terminal major protective region epitope about 0.7kb segment. We expected the recombinant product to bring into full protective immunity with the protecitive antigenic glycoprotein encoded by M segment and the nucleocapsid protein encoded by S segment, so as to settle a solid foundation in the study of gene immunization by recombinant DNA vaccine containing antigenic segments of two serotypes of hantavirus, until we can control the infection caused by two serotypes of hantavirus in the epidemic areas and as a preliminary study for hantavirus DNA vaccine.2, In part II of this study, first of all we applied different doses of recombinant plasmid pEGFP-M-S to BALB/c for gene immunizing by means of intramuscular injection, the specific humoral immunity and cellular immunologic response were detected in the immunized mice. Then the specific humoral and cellullar immunologic response were comparatively studied immunized with two kinds of immunization ways, which is by intranasal immunization or intramuscular injection at the same dose of recombinant DNA vaccine.Methods 1 % Plasmid pUCM which containing Seoul virus M segment l~1678bp was used as a polymerase chain reaction (PCR) template, the targeted 47-1666bp product was cloned into the plasmid pcDNA3.1, and the resulted product is noted as recombinant plasmid Ml; Plasmid pGEM which containing Seoul virus M segment 1601—3638bp was used as a PCR template, the targeted 1661-3445bp product was cloned into the plasmid pcDNA3.1, and the resulted product is noted as recombinant plasmid M2; Plasmids Ml and M2 were digested by BamH I and Kpn I, respectively. We reclaimed the 7.1kb long fragment and the 1.8kb short one, respectively and ligated them in order by T4 DNA ligase, the resulted product was noted as recombinant plasmid pcDNA3.1-M. Plasmid PJSA1175-HVS which containing Hantaan virus 76-118 strain S segment was used as a PCR template, the targeted 40-795bp product was cloned into the plasmid pEGFP, and the resulted product is noted as recombinant plasmid pEGFP-HTNV-S0.7. Plasmids pcDNA3.1-M and pEGFP-HTNV-S0.7 were digested by Xho I and Pst I, respectively. We reclaimed the 3.4kb short fragment and the 5.4kb long one, respectively and ligated them in order by T4 DNA ligase, the resulted 4.1kb product was noted as recombinant plasmid pEGFP-M-S. Plasmid pEGFP-M-S was tranfected into fibroblast and expressed for 48,72h.2s The recombinant plasmid pEGFP-M-S was extracted magnanimously. 3 different doses (lug , 10ug> 50ug) of recombinant plasmid were applied to BALB/c mice by means of quadriceps muscle of thigh intramuscular injection. ELISA (enzyme-linked immunosorbent assay) was used to detect the specific antibody IgG and IgA. The spleen cells of immunized mice were obtained with sterility to analyze the CTL (cytotoxic T lymphocyte) activity. Flow cytometer was used to test the percentage of CD4+and CD8+T lymphocyte. MTT (methyl thiazolyl tetrazolium) method was used to examine the reproductive activity of lymphocytes.3 n Recombinant plasmid pEGFP-M-S was used to immunize mice at a dose of 50ug by 2 different ways, intranasal immunization or quadriceps muscle of thigh intramuscularinjection. ELISA (enzyme-linked immunosorbent assay) was used to detect the specific antibody IgG and IgA. The spleen cells of immunized mice were obtained with sterility to analyze the CTL (cytotoxic T lymphocyte) activity. Flow cytometer was used to test the percentage of CD4+and CD8+T lymphocyte. MTT (methyl thiazolyl tetrazolium) method was used to examine the reproductive activity of lymphocytes.Results 1 ^ We obtained recombinant plasmid Ml, M2, pcDNA3.1-M, pEGFP- HTNV -S0.7 and pEGFP-M-S successfully.2> Recombinant plasmid pEGFP-M-S were applied to BALB/c mice at a dose of lug, lOug and 50\ig respectively by means of quadriceps muscle of thigh intramuscular injection. The mice immunized with lug recombinant plasmid or the control plasmid pEGFP were immunologic response negative; at a dose of 10ug and 50ug recombinant plasmid, the mice were IgG positive at different serum dilution and OD values increased with the immunization dose. The titers of serum specific IgG antibody were all 1: 160. With the increasing immunization times, at a dose of lOug and 50ug recombinant plasmid, the mice were IgA positive in their intestinal juice and there was a significant difference with the control (P<0.05). 2 weeks after the last immunization, the specific cytotoxicity T lymphocyte (CTL) effect to the target cells at a immunization dose of lOug and 50ug increased with the increasing ratio of E/T and there was a significant difference with the control (PO.05), while the immunization groups at a dose of lug or plasmind pEGFP had little CTL effect. The percentage of CD4\ CD8+ T lymphocyte in the spleens of recombinant plasmid immunization groups increased progressively with the increasing immunization doses and immunization times. And the T lymphocytes showed significant multiplication effect after stimulated by ConA, there was a significant difference in the stimulation index with the control (P<0.05).3.. Recombinant plasmid pEGFP-M-S was used to immunize mice at a dose of 50ug by 2 different ways, intranasal immunization or intramuscular injection. The IgG antibodylevel in the intranasal immunization way was lower and the IgA was higher than in the other way, but there was no significant difference between the 2 ways (P>0.05).2 weeks after the last immunization, there was a significant difference between recombinant plasmid immunization groups by 2 different ways and the corresponding control groups in the specific cytotoxicity T lymphocyte (CTL) effect to the target cells with the increasing ratio of E/T, the percentage of CD4+% CD8+ T lymphocyte and the lymphocyte multiplication effect stimulated by ConA (PO.05). But there was no significant difference between the 2 groups(P>0.05).Conclusion K We obtained recombinant plasmid Ml, M2, pcDNA3.1-M, pEGFP-HTNV -S0.7, pEGFP-M-S and the expected 4.1kb recombinant gene, and the recombinant plasmid was expressed transiently in fibroblast L929 successfully 2> The mice immunized with lOug and 50ug recombinant plasmid by intramuscular injection were IgA positive and the titers of serum specific IgG antibody were all 1: 160; the specific cytotoxicity T lymphocyte (CTL) effect to the target cells at a immunization dose of lOug and 50ug increased with increasing ratio of E/T, the CTL effect at the E/T ratio 100 was 25.6 % ±2. 5 % and 62.3 % ±3. 4 %, respectively; recombinant plasmid pEGFP-M-S were applied to BALB/c mice at a dose of lp.g, 10ug and 50\ig respectively by means of intramuscular injection, effective cellular immunologic response was aroused with increasing immunization times and immunization doses; there was comparative humoral and cellular immunologic response at the same dose of recombinant DNA vaccine pEGFP-M-S by means of intranasal immunization or intramuscular injection.