| Aims To express the P30 gene and ROP1 gene of Toxoplasma gondii (Tgondii) in Lactococcus lactis LM 2345 ,respectively. And then observe the potential of recombinant L. lactis vaccine as a effective vaccine against Toxoplasma gondii. Methods Purified parasites were obtained by passage through a column of CF-il cellulose. After isolating total RNA from tachyzoites by AGPC method, poly-A~ RNA was isolated by standard oligo-dT affinity chromatography. The cDNA was synthesized with MMLV reverse transcriptase and digested by sf1. After size-selected by chroma spin-400, the cDNA material was ligated into X TriplEx2 vctor arms, packaged in vitro using extracts and used to infect the host bacteria E. Coli XLL Blue. In order to determine the average length of the cDNA inserts, 16 plaques were randomly picked for PCR screening .Then an DNA fragment of 795 bp encoding the P30 antigen and an DNA fragment of 1249 bp encoding the ROP1 antigen of Tgondii was obtained from the cDNA library by polymerase chain reaction(PCR). The two genes were cloned and the DNA sequences were determined subsequently. The two genes were then introduced into a fusion protein expression vector pGEX-5x-3, and were transformed into BL21 strain cells of E.coli for expression ,respectively. A fusion protein with a molecular weight of 45 kDa(OST-P30) and a fusion protein with a molecular weight of 70 kDa(GST-ROP1) were purified by affinity chromatography, and were analyzed by SDS-polyacrylamide-gel-eletrophoresis(SDS-PAGE) followed by Western blotting and immuno-staining. The BaniHl/XhoI digested fragments of pSK-P30 were inserted into the same site of pskPsT, then introduced into L2-PTRK, a shuttle plasmid between E.coli and L L.actis. The BamHI/XhoI digested, fragments of pSK-ROP1 were inserted into the same site of L2-PTRK-P30. The L2-PTRK -P30 and L2-InPTRK-ROP1 were transformed to Lactococcus lactis LM 2345 by electroporation. The recombinant L Lactis were cultured in GML7 medium overnight The lysate of L Lacti~ were performed by SDS-PAGE and immunoblotting on nitrocellulose membrane according to standard protocols. The recombinant Llactis vaccine were administered orally to BALB/c mice. After 7 doses 4 weeks interval vaccination, mice in every group were challenged on day 49 with 1x102 tachyzoites stage Toxoplasma gondii(RH strain) parasites . The levels of LFN- Y and IL-2 were detected by ELISA. Results The filtration through cellulose columns method produced the better RNA quality. The packageing efficiency were about2.1x 106 pfu/ml(Kunshan strain).. 2.1 x i05 pfu/ml(RH strain). The recombinant rate of two cDNA library were 99% clones/ug cDNA. The average length of cDNA inserted is about 1kb. Comparison of the P30 cDNA sequences between Kunshan strain and RH strain showed that there were only two base pairs different from each other within the 783 bp coding sequence. Comparison of the ROP1 sequence we obtained to the sequence reported by ossorio showed that the gene we obtained contained a insert of 96bp at nt 447 and nt 448 of the gene reported by Dssorio ,there are 14 different nucleotides and 3 different amino acids between the two genes. The identity of the two nucleotide sequence is 91.19%. Distinct protein band with a molecular weight of 56 kDa (GSTP30)and distinct protein band with a molecular weight of 70 kDa (GSTROP1)were detected on SDS-PAGE and its antigenicity were confirmed by immuno-staining . A protein band with a molecular weight of 30 kDa(P30) and a protein band with a molecular weight of 43 kDa(ROP1) can reacted with the antisera from a patient with acute toxoplasmosis, respectively. The survival time was 8.5 days for control group(empty), 14.25 days for p30 group, 7.75 days for ROPI group, 7.5 days for P30+ROP1 group.IvConclusion The cDNA library constructed has large content and good quality. Expression of P30 and ROP1 were achieved in L Lactis. The oral delivery of recombinant L.lactis vaccine has potential as a effective vaccine against Toxoplasma gondii.Yang Qiulin... |
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