ABSTRACT
Objective To clone and express surface antigen SAG4 gene of Toxoplasma gondii, and analyze its immunoreactivity. Methods Specific primers were designed based on the reported SAG4 gene of T. gondii RH strain (GenBank Accession No: AF340224.1). Using genomic DNA from T. gondii as templates, SAG4 gene was amplified by PCR. The PCR product was cloned into pMD19-T vector and identified by digestion with restriction enzyme and PCR. Then the target fragment was subcloned into pET28a(+) vector, transformed into E. coli BL21 and followed by expression of the protein induced by IPTG. The protein was identified by Western blotting. Results The target gene was amplified with the length of 537 bp. Sequence analysis showed that the predicted amino acid sequence was identical with that of SAG4 as a membrane protein in T. gondii. After induced by IPTG, the recombinant SAG4 protein existed in an inclusion body form. The recombinant SAG4 (Mr 18 740) was recognized by serum of infected mice. Conclusion SAG4 has been expressed and shows certain immuno-response activity.
ABSTRACT
Objective To observe the effect of different cryoprotective agents and temperature factors on the viability of Blastocystis hominis so as to explore the ideal method for preservation of B.hominis.Methods B.hominis agents were obtained from a patient's fecal specimen.Having washed by normal saline and divided into tubes,the samples were cryopreserved in-20 ℃ refrigerator or in-l96 ℃ liquid nitrogen with 10% DMSO,40% glycerol and 15% ethylene glycol respectively.The thawed B.hominis agents were then used for culture.By trypan blue staining and microscopy,the viability and proliferation of those resuscitative cells were investigated.Results B.hominis survived for 3 weeks at 18 ℃-20 ℃ while less than 1 week at 4 ℃-6 ℃.When stored in-20 ℃ refrigerator or liquid nitrogen with cryoprotective agents,they survived for more than 3 months.The cryopreservation with 40% glycerol at-196 ℃ for 6 months resulted in 41.7% viability of the revivified cells.Cleavage cells were easily observed after culturing for 72 hours.Conclusion Preserving B.hominis in liquid nitrogen with 40% glycerol is an optimal cryopreservation protocol.
ABSTRACT
Anopheles minimus collected from Yuanjiang, Yunnan Province, were bred with standard methods in lab. The ovarian nurse cells of A.minimus were separated and stained, and the whole polytene chromosomes were photographed under light microscope and compared with A.minimus from Guangxi. 365 samples of ovarian nurse cells were observed. The chromosomes included one telocentric sex-chromosome X, two submetacentric autosomes II(autosome II right arm, 2R and autosome II left arm, 2L) and two metacentric autosomes III(autosome III right arm, 3R, and autosome III left arm, 3L). The X is the shortest chromosome and the 2R is the longest one. In comparison with the pattern of polytene chromosomes of A. minimus from Guangxi, difference at 12 positions has been found at the parts of arms in banding sequences.