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Cloning and Expression of SAG5B and SAG1 of Toxoplasma Gondii and Their Immunoprotective Role in Mice Against T.gondii

Author: QiaoZengPei
Tutor: ShenJiLong
School: Anhui Medical University,
Course: Pathogen Biology
Keywords: Toxoplasma Vaccine Clone Expression Protective Immunity
CLC: R392
Type: Master's thesis
Year: 2008
Downloads: 90
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Abstract


Objective: To clone Toxoplasma gondii the the the RH strain sheet film sag1 as well as a newly discovered gene sag5b, and the building prokaryotic expression vector pET28a/sag1 and the pET28a/sag5b, expression of of Toxoplasma surface membrane protein SAG1 and SAG5B of. The recombinant protein immunized mice, the ability to observe the mice against Toxoplasma gondii infection. Comparison RH strain of Toxoplasma gondii genome with Praugniud strains Toxoplasma gondii genome sag5b of genes for the possibility of the identification of the virulence of the strains of insects. : Recovery in our laboratory liquid nitrogen species preservation RH strain Toxoplasma gondii tachyzoites, Balb / c mice were inoculated intraperitoneally transferred species generation culdocentesis collection, purification of Toxoplasma gondii, and proteinase K lysis method to extract genomic DNA, while crude preparation of tachyzoites antigen to immunize New Zealand white rabbits, collecting rabbit antiserum against Toxoplasma gondii. Killed and the brain tissue of mice containing Praugniud strains of Toxoplasma cysts obtain brain tissue containing cysts. Respectively at both ends of the primer in the primer design and introduction of EcoRI and XhoI, and EcoRI and HindIII digestion sites. PCR amplification to Toxoplasma sag5b sag1 gene fragment, target gene sag5b and sag1 gene fragment was inserted into the cloning vector pGEM-T to extract the recombinant plasmid, double digestion and target gene, inserted into the prokaryotic expression vector pET28a recombinant dual enzyme cut, PCR and sequencing, and transformed into E. coli E.coli BL21 (DE3) and IPTG induction. Affinity chromatography purification of recombinant proteins, SDS-PAGE and Western blotting to verify expression levels and immune activity and the the purified rSAG5B and rSAG1 protein concentration was determined by Lowry's method. The two purified proteins, as well as Toxoplasma crude antigen were subcutaneously immunized Balb / c mice, and then attack the infection, in order to observe the survival of mice. To adjuvant blank. Results: PCR from T. gondii RH strain genomic clone out of the 1104bp of sag5b target gene fragment. Successfully cloned into pET28a. the sag5b-pET28a by EcoRI and XhoI double digestion, consistent with the size of the target gene gene fragment sequencing results with GenBank homology than sag5b 100%. Host bacteria containing sag5b-pET28a E.coliBL21 (DE3) after IPTG induction efficient express rSAG5B protein. RH strain of Toxoplasma gondii genome and the brain tissue cysts containing Praugniud strains of Toxoplasma as a template, PCR amplified by sag5b, the gene is only found in the genome of the RH strain. In addition, using PCR out the 1101bp of sag1 objective gene fragment was cloned from the genome of T. gondii RH strain. Cloned into pET28a vector. sag1-pET28a with EcoRI and HindIII double digestion, consistent with a gene fragment obtained with the gene size sequencing results with GenBank than sag1 100% homology. Efficient expression host strain containing sag1-pET28a E.coliBL21 (DE3) after IPTG induction rSAG1. These two recombinant protein was purified by Ni2 affinity chromatography purification the of high purity rSAG5B and rSAG1 protein. SDS-PAGE analysis of its molecular weight: rSAG5B 43 KDa rSAG1 to 30 kDa, both with their respective expected molecular size match. The Western blotting Show: the corresponding antiserum rSAG5B with rSAG1 protein can be anti-Toxoplasma antibodies identify two purified recombinant proteins with a specific immune response. These two purified recombinant proteins, and Toxoplasma crude antigen preparation subcutaneously immunized Balb / c mice with Freund's adjuvant, Freund's adjuvant at the same time as the blank control. Each mouse antigen used in an amount of 20μg, strengthening after two weeks time, the adjuvant changed to Freund's incomplete adjuvant. One month after the last immunization, intraperitoneal injection of mice to challenge infection with T. gondii RH strain tachyzoites statistical comparison the rSAG5B and rSAG1 crude antigen immune protective effect on mice there was no significant difference (P gt; 0.05) long survival time than those in the control group (not immunohistochemistry), there is a significant difference (P lt; 0.05). Conclusion: Toxoplasma gondii RH strain genomic acquired sag5b gene as well sag1 of to build a sag5b-pET28a/sag1-pET28a recombinant plasmid and was highly expressed; contrast RH strain genome shows that with Praugniud strain genome, sag5b might as identification strong strains with an attenuated strain of genetic markers. Preparation of RH strain of Toxoplasma gondii crude antigen and immunize New Zealand white rabbit antiserum against Toxoplasma gondii. The crude antigen purified recombinant protein were used to immunize Balb / c mice, and compared with blank, rSAG5B and rSAG1 mice immune protection, and is similar to the role of the crude antigen. Research of Toxoplasma gondii new surface membrane protein found SAG5B protective immunity in mice, to help develop effective preventive vaccine of Toxoplasma gondii infection.

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