PCR clone of novel Staphylokinase gene from Staphylococcus aureus

S450 Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576

[P-M.64]

Influence of Endosulfan on Allergic Response in Mouse

Sogo Nishimoto 1,2,∗, Satoko Atobe 2, Masaaki Okabe 1,2, KoichiAkiyama 3, Yoshimi Kakinuma 2,3, Takuya Sugahara 2,4

1 Center for Marine Environmental Studies, Ehime University, Japan2 Faculty of Agriculture, Ehime University, Japan3 Integrated Center of Science, Ehime University, Japan4 Southern Ehime Fisheries Research Center, Ehime University, JapanKeywords: Endosulfan; Allergy; Immune response; Cytokine

Many scientists have strong interest in studying the effectsof agricultural chemicals on our health. We focused on endosul-fan, one of the agricultural chemicals, because the chemical waslisted as new POPs candidates in 2005 (Stockholm Convention in2005 “New POPs”). There are many reports concerning the poi-sonous effects of endosulfan on reproductive organs (Naqvi andVaishnavi, 1993). However, there are only few reports on evaluationof immune toxicity of endosulfan. Hence, we investigated the effectof �-endosulfan (one of the isomers) on immune system, especiallyallergy response.

�-endosulfan was orally administrated to ovalbumin (OVA)-challenged allergy mice for 27 days. Blood and spleen werecollected on day 28, and spleen lymphocytes were isolated and cul-tured for 48 h. OVA-specific Ig levels and cytokine levels in serumand culture supernatant of spleen lymphocytes were measuredby enzyme-linked immunosorbent assay (ELISA). The effect of �-endosulfan on allergen-induced degranulation by rat basophilicleukemia cell line RBL-2H3 cells was examined.

Oral administration of �-endosulfan increased OVA-specific IgEand IgG1 levels in serum. �-endosulfan also enhanced interleukin(IL)-4 level in serum, and IL-4 production by spleen lymphocytes. IL-4 is a cytokine inducing IgE production by B cells. On the other hand,interferon (IFN)-� level in serum was not affected by intake of �-endosulfan. Allergy response is potentiated by imbalance betweenhelper T sub-populations, Th1 and Th2 (Abbas et al., 1996). �-endosulfan activated IL-4-producing Th2 cells. This suggests that�-endosulfan inclines Th balance toward Th2 dominance, andallergy response is facilitated by imbalance of Th1/Th2. In addition,�-endosulfan activated degranulation of RBL-2H3 cells. This resultmeans that �-endosulfan exaggerates allergy symptoms mediatedby histamine and leukotriene in granules. Our findings suggest thatintake of �-endosulfan is a serious risk for our health.

Acknowledgement

This work was supported by Grant-in-Aid for Young Scientists(B) from the Ministry of Education, Science, Sports, and Culture inJapan.

References

Naqvi, S.M., Vaishnavi, C., 1993. Bioaccumulative potential and toxicity of endosulfaninsecticide to non-target animals. Comp. Biochem. Physiol. C. 105, 347–361.

Abbas, A.K., et al., 1996. Functional diversity of helper T lymphocytes. Nature 383,787–793.

doi:10.1016/j.jbiotec.2010.09.652

[P-M.65]

PCR clone of novel Staphylokinase gene from Staphylococcusaureus

Abolfath Ebrahimi ∗, Abdollah Ghasemian, Younes Ghasemi

Department of Pharmaceutical Biotechnology, Faculty of Pharmacyand Pharmaceutical Sciences Research Center, Shiraz University ofMedical Sciences, Shiraz, Islamic Republic of IranKeywords: Gene cloning; Staphylokinase; Staphylococcus aureus;E.coli BL21(DE3)

Introduction: Staphylokinase (SAK) is an extracellular protein.It is a potential therapeutic thrombolytic protein which convertsthe plasminogen, the inactive proenzyme of the fibrinolytic sys-tem, into plasmin. It is supposed to be effective in acute myocardialinfarctions and it has a lower antigenicity, higher fibrin-specifity,and greater efficacy in comparison with streptokinase

Methods: In the present work, the Staphylokinase from Staphy-lococcus aureus PTCC 1112 was cloned in pET15b and expressed inE.coli. Total genomic DNA were isolated and used for PCR amplifi-cation of the SAK gene. Desired sequence was amplified using thespecific primers, which amplify a ∼500-bp of the structural SAKgene. The resulting PCR amplicon was ligated in the E.coli expres-sion vector pET15b. E.coli BL21(DE3) cells were transformed withthe recombinant plasmid to get the expression strain

Results: The amplified sequence was verified by DNA sequenc-ing and compared with other SAK genes on GenBank using BLASTtools. The staphylokinase activity was measured by using humanplasminogen coupled chromogenic substrate assay by measuringthe absorbance of released para-nitroaniline from S-2251 (D-Val-Leu-Lys-4-nitroanilide) at 405 nm. The recombinant enzyme hasthe same activity as wild-type staphylokinase. Molecular weight ofrecombinant protein estimated to be ∼ 15 kDa by SDS-PAGE

Discussion: The characterization of new recombinant staphy-lokinase and the development of rapid, simple and effectiveproduction methods are not only of academic interest but also ofclinical importance

doi:10.1016/j.jbiotec.2010.09.653

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Preparation of chitosan/carboxymethyl dextran nanoparticlesby polyelectrolyte complexation

E.J. Lee 1, S.A. Khan 2, Y.-B. Kim 2, K.-H. Lim 2,∗

1 Kyungpook National University, Republic of Korea2 Daegu University, Republic of KoreaKeywords: Chitosan; Carboxymethyl dextran; Nanoparticle; Poly-electrolyte complexation

The optimum condition was pursued for the preparation of thechitosan (polycation) /carboxymethyl dextran (CMD) (polyanion)polyelectrolyte-complex. Both the various mass-ratios of chitosanand CMD and the various pH of used chitosan solution were investi-gated to evaluate their effect on the turbidity of dispersion, the yieldof dried mass, and the particle-size and the chemical structure oftheir polyelectrolyte-complex. The investigation was performed bythe measurement of absorbance, the observation by SEM and theanalysis of FT-IR spectrum. It was found that no turbidity appearedat all the mass-ratios studied with the chitosan solution of pH 2.With the chitosan solution of pH 5, a marked turbidity with no vis-ible aggregates was obtained at the chitosan-CMD mass-ratio of1:4. However, at the same pH, the mass-ratio of 1:5 led to the for-