Table of Content

28 February 2013, Volume 31 Issue 1

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Immune Response in Mice Induced by Complex Gene Vaccine pcSAG1-ROP5 of Toxoplasma gondii

ZHAO Huan-ge，FAN Zhi-gang，HUANG Feng-ying，HUANG Yong-hao，

2013, 31(1):  1-1-5. 

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Objective  To observe the immune response induced by complex gene vaccine pcSAG1-ROP5 of Toxoplasma gondii in mice.  Methods  The recombinant eukaryotic expression plasmids pcSAG1, pcROP5 and pcSAG1-ROP5 were constructed and identified by PCR, restriction enzyme digestion, and sequencing. The three recombinant plasmids were transfected into HeLa cells to express in vitro and identified by Western blotting analysis. Seventy Kunming mice were randomly divided into 5 groups with 14 each, i.e. pcSAG1 group, pcROP5 group, pcSAG1-ROP5 group, blank plasmid group and PBS control group. The mice were immunized intramuscularly with pcSAG1, pcROP5, pcSAG1-ROP5, pcDNA3.1, and PBS, respectively, every two weeks for three times. Sera were collected before each injection and 2 weeks after the last immunization. The titer of mice serum in pcSAG1-ROP5 group combined with recombinant protein SAG1, ROP5 and SAG1-ROP5 and the level of IgG against T. gondii in 5 groups were determined by ELISA. Three weeks after the last immunization, ten mice of each group were challenged with 103 tachyzoites of the virulent T. gondii RH strain to observe the survival time. One week later, the rest four mice in each group were sacrificed and the supernant of cultured splenocytes was collected for the detection of IFN-γ and IL-4.  Results  Western blotting showed that the recombinant plasmids pcSAG1, pcROP5 and pcSAG1?鄄ROP5 were expressed in HeLa cells with Mr 31 000, 57 000, and 88 000, respectively. The serum titer in pcSAG1-ROP5 group combined with SAG1, ROP5 and SAG1-ROP5 was 1 ∶ 320, 1 ∶ 160, and 1 ∶ 2 560, respectively. The IgG level kept rising in pcSAG1, pcROP5 and pcSAG1-ROP5 groups. Two weeks after the last immunization, the IgG level in pcSAG1-ROP5 group was higher than those in other groups（P<0.05）. After a lethal challenge of T. gondii RH strain, the survival time of the mice in pcSAG1-ROP5 group was （288±7） h, which was 48 h and 96 h longer than the groups of pcSAG1 and pcROP5, respectively （P<0.05）. Four weeks after the last immunization, IFN?-γ in splenocyte culture of pcSAG1-ROP5 group ［（908.52±6.31） pg/ml］ was higher than other groups （P<0.05）, with no significant difference in IL-4 （P>0.05）.  Conclusion  Compared with the single gene vaccines pcSAG1 and pcROP5, higher levels of IgG and IFN-γ and longer survival time are observed in mice immunized with pcSAG1-ROP5.

Separation and Identification of the Surface and Secreted Proteins in Toxoplasma gondii by Biotin Labeling and Proteomics Methods

LIU Yuan1，XUE Feng1，2，HUANG Min-jun1，2，GAN Shao-bo1，2，GU Jun-chao1，2 *

2013, 31(1):  2-6-11. 

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Objective  To separate and identify the surface proteins and secreted proteins of Toxoplasma gondii tachyzoties of RH strain.  Methods  T. gondii tachyzoties were cultured in Vero cells, and purified by filtration and Percoll cell separation solution. The biotin-labeled tachyzoites were lysed, and the surface and secreted proteins were separated by NeutrAvidin agarose beads. After condensation and SDS-PAGE, the protein were collected, digested and identified by LC/MS-MS.  Results  A total of 785 T. gondii proteins were identified, 81 (10.3%) of which were originally annotated as the surface or secreted proteins. Among the highly-expressed (PSM>10) 65 proteins, 43 (66%) were originally annotated as surface or secreted proteins, while the others were predicted unknown proteins.  Conclusion  The surface and secreted proteins of T. gondii are separated by biotin labeling and avidin chromatography, among which some potential new surface or secreted proteins of T. gondii are identified.

Cloning，Expression and Immunogenicity Analysis of Malate Dehydrogenase Gene of Toxoplasma gondii

LIU Zhuan-zhuan1，2，YANG Yan-ping1，2，YIN Guo-rong 2 *，WANG Hai-long 2，

2013, 31(1):  3-12-17. 

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Objective  To clone and express the malate dehydrogenase（MDH） gene of Toxoplasma gondii，　and analyze the immunogenicity.  Methods  Total RNA was extracted from tachyzoites of RH strain of T. gondii（GenBank accession No. AY650028）. The coding region of TgMDH was amplified with a pair of specific primers. The product of RT?鄄PCR was digested with double restriction enzyme and ligated into pET30a（+） vector. The recombinant pET30a（+）-TgMDH plasmid was transformed into E. coli DH5α. The positive clones were confirmed by the double restriction enzyme digestion， PCR and sequencing. The correct plasmid was transformed into E. coli BL21 and induced by IPTG. The expressed proteins were analyzed by SDS-PAGE. Conditions for expression were optimized. Abundant soluble rTgMDH protein was purified with Ni-NTA affinity chromatography. Mice was intranasally immunized with purified rTgMDH and murine anti-rTgMDH serum was prepared. Western blotting with murine anti-rTgMDH serum and rabbit anti-T. gondii serum was used to analyze its immunogenicity.　 Results 　The product of RT-PCR was with 951 bp. The recombinant plasmid pET30a（+）?-TgMDH was confirmed by the double restriction enzyme digestion，　PCR and sequencing. A soluble recombinant protein with relative molecular weight of 36 000 was analyzed by SDS-PAGE，　followed by coomassie blue staining. Western blotting revealed that rTgMDH can be recognized by murine anti-rTgMDH serum and rabbit anti-T.  gondii serum. 　Conclusion　 TgMDH gene has been expressed in prokaryotic expression system and shows immunogenicity.

Screening of Host Cell Proteins that Interact with Toxoplasma gondii ROP18 via Yeast Two-hybrid System

DU Jian 1 *，AN Ran 1，CHENG Li 1，CHEN Ying 2，SHEN Ji-long 3

2013, 31(1):  4-18-22. 

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Objective  To screen the host cell proteins that can interact with Toxoplasma gondii ROP18 by using yeast two-Hybrid system.  Methods  The ROP18 gene fragments were amplified by RT-PCR from mRNA of T. gondii RH strain. The product of RT-PCR was digested with double restriction enzyme and was subcloned into the bait vector pGBKT7. The recombinant plasmid was transferred into yeast AH109 strain. Its toxicity and the autonomous activating activity were tested. The human fetal brain cDNA library was screened with pGBKT7-ROP1825-251aa as the bait plasmid by yeast two-hybrid system.  Results  The bait was constructed and AH109/PGBKT7-ROP18 showed an autonomous activity. The yeast strain AH109/pGBKT7-ROP1825-251aa line was then mated with the Mate & PlateTM Human Fetal Brain cDNA library. Using the selection procedures， eight novel host cell proteins were obtained： damage-specific DNA binding protein 1（DDB1）， torsin A interacting protein 1（TOR1AIP1）， integrin beta 1， solute carrier family 3 （SLC3A2）， tyrosylprotein sulfotransferase（TPST2）， OCIA domain containing 1（OCIAD1）， Der1-like domain family member 2（DERL2）, in addition to Homo sapiens activating transcription factor 6 beta（ATF6）.  Conclusion  Eight novel host cell proteins have been obtained via yeast two-hybrid system, which can interact with TgROP18.

Prokaryotic Expression and Histolocalization of the Taenia solium CDC37 Gene

HUANG Jiang1， 2，LI Bo1，DAI Jia-lin1，ZHANG Ai-hua2 *

2013, 31(1):  5-23-26. 

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Objective  To express Taenia solium gene encoding cell division cycle 37 protein（TsCDC37） and investigate its antigenicity and localization in adults of Taenia solium.  Methods  The complete coding sequence of TsCDC37 was amplified by PCR based on the recombinant plasmid clone from the cDNA library of adult Taenia solium. The PCR product was cloned into a prokaryotic expression vector pET-28a（+）. The recombinant expression plasmid was identified by PCR， double endonuclease digestion and sequencing. The recombinant plasmid was transformed into E. coli BL21/DE3 and followed by expression of the protein induced by IPTG. The mice were immunized subcutaneously with purified recombinant TsCDC37 formulated in Freund’s adjuvant. The antigenicity of the recombinant protein was examined by Western blotting. The localization of TsCDC37 in adult worms was demonstrated by immunofluorescent technique.  Results  The recombinant expression vector was constructed successfully. The recombinant protein was about Mr 52 000， it was then purified and specifically recognized by immunosera of SD rats and sera from patients infected with Taenia solium， Taenia saginata or Taenia asiatica. The immunofluorescence assay revealed that TsCDC37 located at the tegument of T. solium adult and the eggs.  Conclusion  TsCDC37 gene has been expressed with immunoreactivity. The recombinant protein is mainly expressed in tegument and egg， and is a common antigen of the three human taenia cestodes.

Screening and Analysis of Peptides Specifically Binding to the Schistosomulum Tegument of Schistosoma japonicum

LIU Yan1，ZHANG Zu-ping2，WANG Ke-geng1，GU Kong-zheng2，CAI Li-ting2，ZENG Qing-ren2 *

2013, 31(1):  6-27-32. 

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Objective  To screen and analyze the peptides in 12 phage-display peptide library specifically binding to the schistosomulum, not cercaria, tegument of Schistosoma japonicum.  Methods  A 12 phage-display peptide library was screened with the S. japonicum schistosomula and cercariae as the target cells for biopanning by degrees, 15 positive clones were picked randomly and deduced by DNA sequencing. According the sequencing result, ELISA test, elution recovery test and immunohistochemical staining were performed to determine the specificity of the phages to the tegument. To further examine its binding properties, the positive peptide conjugated to RhB and recombinant pEGFP?-C2 plasmid were similarly synthesized.  Results  After 3 rounds of biopanning, the phage recovery rate increased from 3.50×10-5% to 3.20×10-2%, indicating that the phage library was successfully enriched in the tegument of schistosomula. The analyzed sequences were identical with 3 peptide sequence of ZL6, ZL4 and ZL1. ELISA showed that the P/N value of MppZL4, MppZL6 and MppZL binding the schistosomulum membrane protein was 6.72，3.65 and 2.22, while 1.58，5.15 and 1.20 of binding the membrane protein of cercariae, respectively. Elution recovery test showed that the elution recovery rate of MppZL4 ［（4.60±0.27）×10-2%］ was much higher than that of MppZL6 ［（2.10±0.23）×10-3%］, MppZL1 ［（1.20±0.28）×10-3%］ and M13KE［（1.30±0.60）×10-7%］（P<0.01）. Immunohistochemical staining showed that MppZL4 specifically bound to the tegument of schistosomula with a positive rate of 83.0%（83/100）. Fluorescent microscopy revealed that the synthesized RhB-ZL4 bound to the tegument of schistosomula. The ZL4/pEGFP-C2 plasmid was introduced into juvenile S. japonicum and expressed in the parasite.  Conclusion  The peptide of ZL4 specifically binds to the schistosomulum tegument but not to that of cercaria.

The Binding Property of Different Regions in Duffy-binding-like Domain α of Plasmodium falciparum Erythrocyte Membrane Protein 1 to Heparin

ZHANG Yan1，SUN Xi-dong2，YANG Chun3，ZHANG Ya-na1，

2013, 31(1):  7-33-38. 

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Objective  To clone and express Plasmodium falciparum erythrocyte membrane protein 1 DBLα （PfEMP1-DBLα） and three fragments genes, and screen the strongest affinity sequence with the red blood cell surface receptors?鄄heparin or heparin sulfate in the structure of PfEMP1-DBLα.  Methods  The sequence of PfEMP1-DBLα1245 was optimized according to the characteristics of E. coli codon, synthesized, and divided into three fragments （DBLαA, DBLαB, and DBLαC） by PCR. Full-length gene and three gene fragments were subcloned into PGEX-4T-1 vector, and transformed into E. coli BL21 and then induced with IPTG for expression. The recombinant protein was purified from bacterial lysates using gluthathione-Sepharose 4B. Heparin affinity test and glycosaminoglycan (GAG) inhibition test were used to analyze the affinity between recombinant protein and heparin.  Results  Four recombinant proteins（DBLα1245, DBLαA, DBLαB, and DBLαC） were expressed as solubility and the relative molecular weight （Mr 73 600, Mr 41 600, Mr 42 500, and Mr 41 500） were conformed to the prediction size. Heparin affinity test and GAG inhibition test showed that the four recombinant proteins were binded to the heparin-Sepharose, but not for the GST control. DBLαC (Q285-Y415) had the strongest affinity to heparin.  Conclusion  The strongest affinity sequence with heparin or heparin sulfate in the structure of PfEMP1?鄄DBLα is Q285-Y415, which plays a role in binding of Plasmodium falciparum infected red blood cells to the peripheral red blood cells.

Ａ New Species of the Genus Nanhaipotamon（Decapoda ∶ Potamidae） Serving as Intermediate Host of Paragonimus skrjabini

LIN Guo-hua1，CHENG You-zhu2 *，CHEN Shao-hong3

2013, 31(1):  8-39-42. 

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Objective  To describe a new species of the genus Nanhaipotamon.  Methods  Freshwater crabs were collected in the counties of Yongtai, Minqing, Youxi, Songxi, Zhenghe and Shouning, Fujian Province. The morphological characteristics of the crabs were described. The habitats were observed and crabs examined for the presence of Paragonimus metacerariae.  Results  Ａ new species of freshwater crabs named as Nanhaipotamon fujianense sp. nov. was described: holotype（FJ6132-1）: ♂, carapace length 18.44 mm, breadth 23.64 mm, thickness 12.61 mm; allotype（FJ6132-2）: ♀, length 18.76 mm, breadth 25.25 mm, thicknes 14.31 mm, collected from Yongtai County in the middle of Fujian （N 25°44,778′; E118°32,278′, and 232 m above sea lever）. Distal segment of the first male pleopod with triangle convex inner-distal angle, and the axe-like expanded out-distal angle. The out-lateral border slightly sloped downwards. The segment length is 2.1 times as long as the subdistal segment. The crabs usually lived in the crevice of small stream. Paragonimus metacerariae were found in the crabs collected from Yongtai, Minqing, Youxi, Songxi and Zhenghe Counties.   Conclusion  Ａ new species of freshwater crab（Nanhaipotamon fujianense sp. nov.） has been recorded which serves as the intermediate hosts of Paragonimus skrjabini.

Analysis of Pfcrt Gene Polymorphism in Plasmodium falciparum from Imported Cases

ZHOU Shui-mao, YANG Yan, WU Kai, CHEN Zhi, XU Ming-xing, LIU Yong, WANG Chong-xin

2013, 31(1):  9-43-45. 

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Objective  To identify the incidence of the K76T mutation in Pfcrt gene of imported Plasmodium falciparum and study the Pfcrt gene polymorphism in Plasmodium falciparum.  Methods  Seventy?鄄two blood samples were collected from patients infected with P. falciparum returning from Africa（Nigeria, Equatorial Guinea, Congo, Liberia, Angora and Mali） and Southeast Asia （Myanmar and Indonesia） from 2008 to 2012. According to Pfcrt gene sequence of P. falciparum, nested PCR primers were designed, and the reaction was applied with P. falciparum DNA in the blood samples as templates. PCR products were identified by Apo I digestion.  Results  Among 72 blood samples of P. falciparum, mutant Pfcrt alleles were found in 41 samples（57.7%, 41/71） and wild type Pfcrt alleles were found in 30 samples （42.3%, 30/71）. There were 25 samples（50%, 25/50） each with mutant Pfcrt alleles or wild type that were from Africa, while 16 samples（76.2%, 16/21） with mutant Pfcrt alleles and 5 samples （23.8%, 5/21） with wild type that were from Southeast Asia, repectively.  Conclusion  The incidence of Pfcrt gene mutation is different in P. falciparum isolates from different regions.

Immunogenicity Analysis of Recombinant GST Protein of Fasciola hepatica in SD Rats

WEN Xiao-bo，RAN Xu-hua*，WANG Chun-ren，SONG Bai-fen，WEI Xiao-man，

2013, 31(1):  10-46-48,53. 

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Objective  To analyze the immunogenicity of recombinant glutathione S-transferase protein of Fasciola hepatica（FhGST） in SD rats.  Methods  The recombinant expression plasmid pET30a-FhGST was transformed into E. coli BL21（DE3） cells and induced with IPTG for protein expression. The recombinant protein FhGST was analyzed by SDS-PAGE and identified by Western blotting. Twenty SD rats were randomly divided into two groups: immunized group and adjuvant control group. SD rats in immunized group were injected subcutaneously with 200 μg of purified FhGST protein. The adjuvant control group with 10 SD rats received only adjuvants emulsified with PBS. All the rats received three immunizations at 3-week intervals. Serum samples were collected at pre-immunization, the day after each immunization, 3 weeks and 6 weeks after the final immunization. The IgG antibody of rats’ sera was examined by indirect ELISA and spleen lymphocyte proliferation （SLP） was tested by MTT.  Results  The molecular weight of purified FhGST was about Mr 31 300. The recombinant FhGST was recognized by pool sera of goats naturally infected with F. hepatica. The recombinant protein induced specific antibody IgG against GST protein in SD rats significantly higher than that of the control, and the antibody titer reached the peak at 9 weeks after the first immunization（GMT 1 ∶ 89 144）. FhGST protein significantly enhanced the growth and proliferation of rat splenocytes.  Conclusion  The recombinant FhGST protein induces specific immune response in SD rats.

Relationship between Snails and Recent Water Level in Differdent Marshlands in Xingzi County，Jiangxi Province

2013, 31(1):  11-49-53. 

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Objective  To study the longitudinal change of data on Oncomelania hupensis surveillance in different marshlands and the impact of recent water level in Xingzi County, Jiangxi Province.  Methods  All information including water level of hydrometric station and the data of snails at the marshlands of Xiguanhu, Majiawan and Ximiaoqian was collected to explore the longitudinal change of snails and analyze the relationship between snail distribution and recent water level with Spearman rank correlation analysis.  Results  The highest proportion of frames with living snails and living snail densities at Majiawan and Ximiaoqian was 89.66% （442/493） in 2002 and 66.72% （872/1 307） in 2007, 8.33 in 2001 and 7.39 snails per frame in 2006, respectively, and the lowest was 13.26% （126/950） in 2010 and 4.60% （55/1 195） in 2005, 0.42 in 2010 and 0.22 snails per frame in 2002, respectively, and tended to decrease gradually after 2007. At Majiawan,  infected snails were found in 2005 and 2009, the density and proportion of infected snails were 0.0033 and 0.0025 snails per frame, 0.09% （3/3 306） and 0.22% （3/1 389）. Infected snails were found in Ximiaoqian in 2001, 2003, 2005 and 2009, the highest density and proportion of infected snails were 0.005 0 snails per frame and 0.88% （6/684） in 2005. Infected snails were found in Xiguanhu in 2002 and 2003 with a density and proportion of 0.002 9 and 0.002 7 snails per frame, 0.10% （1/974） and 0.32% （1/312）, respectively. The correlation analysis between proportion of frames with living snails and density at Xiguanhu with the average water level of the first and second month before snail survey showed statistical significance, the correlation coefficient was 0.76, 0.71, 0.82 and 0.78（P<0.05）, respectively. The correlation between proportion of frames with living snails and density at Majiawan showed no statistical significance with the average water level of recent three months before snail survey. The proportion of frames with living snails and density at Xiguanhu were negatively correlated with the average water level of the first and second month before snail survey, the correlation coefficient was -0.67, -0.75, -0.79 and -0.72（P<0.05）, respectively.  Conclusion  The change trend of snail indicators in different marshlands in the County and impact of water level in recent three months on snail population are both different, and the snail control strategy in marshlands should therefore be adjusted.

Analysis of Larval Echinococcosis Cases from the National Web-based Infectious Diseases Report System in China in 2011

LI Jun-jian，CHEN Hai-tang，WU Wei-ping*

2013, 31(1):  12-54-56,63. 

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Objective  To analyze the relevant information of echinococcosis cases from the National Web-based Infectious Diseases Report System in China in 2011.  Methods  Data of echinococcosis in 2011 were collected from the Report System of the Chinese Center for Disease Control and Prevention. SPSS 16.0 software was used to analyze the data.  Results  A total of 3 225 cases were reported in 2011, including 1 death and 3 013 effective cases. The three provinces （autonomous region） with high incidence were Xinjiang（occupying 41.5%， 1 251/3 013）, Gansu（16.9%， 509/3 013） and Qinghai（12.0%， 363/3 013）. Cases distributed in all age groups, with the highest incidence in the group of 31-40-year-old. Male to female ratio was 1 ∶ 1.01. The incidence of farmers and herdsmen was highest.  Conclusion  In 2011， the reported cases are mainly distributed in Xinjiang, Qinghai, Inner Mongolia, Gansu, Ningxia, Sichuan and Tibet. Echinococcosis is mainly prevalent in western China.

Rapid Risk Assessment on the Import of American Trypanosomiasis to China

QIAN Ying-jun, LI Shi-zhu, WANG Qiang, ZHANG Li, LIU Wei, CHEN Jia-xu,

2013, 31(1):  13-57-59. 

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American trypanosomiasis, as one of the “neglected tropical diseases”, is a zoonosis induced by Trypanosoma cruzi. It is endemic in 18 countries in the Central and South America, especially in rural areas. A rapid risk assessment was carried out to analyze the potential threat of imported cases to China, which would provide information to policy makers in health authorities.

Advances in Research on the MAPK Signal Transduction Pathway of Echinococcus

WANG Cheng-hua1，LV Hai-long2，JIANG Yu-feng 1 *，PENG Xin-yu2，ZHANG Jing1

2013, 31(1):  14-60-63. 

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Mitogen-activated protein kinase（MAPK） is an important signaling transduction molecules, which can enter the nucleus and activate target gene when it was stimulated and become phosphorylation. MAPK signaling pathway is closely associated with various diseases. Recent studies have indicated that MAPK signaling transduction pathway is also involved in the growth and development of Echinococcus. This review summarizes the progress on the relationship between MAPK signal pathway and Echinococcus.

Advance of Proteomic Research on Schistosome

XU Hong1,2，GUAN Fei1，LIU Wen-qi1 *

2013, 31(1):  15-64-67. 

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Schistosome proteome research may greatly enhance the understanding of immune mechanism, exploration of new diagnostic and vaccine candidates, and the development of new drugs. This article reviews the progress of proteomic research on schistosome from different life-cycle stages.

Preparation of Paraffin Section of Oncomelania hupensis  Cerebral Ganglions

TAN Ping*，ZHANG Jie，LI Qing

2013, 31(1):  16-16-17. 

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Following the method used in other mollusks, the specimen of the Oncomelania hupensis cerebral ganglions was fixed, dehydrated, transparentized, embedded in paraffin， sectioned, and stained with haematoxylin?鄄eosin. The histological structure of the snail cerebral ganglions was clear in the permanent specimen under the microscope.

In vitro Metabolism of Fenbendazole Prodrug

WEN Ai-dan，DUAN Li-ping，LIU Cong-shan，TAO Yi，XUE Jian，WU Ning-bo，

2013, 31(1):  17-68-70. 

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Synthesized fenbendazole prodrug N-methoxycarbonyl-N′-（2-nitro-4-phenylthiophenyl） thiourea （MPT） was analyzed in vitro in artificial gastric juice, intestinal juice and mouse liver homogenate model by using HPLC method, and metabolic curve was then generated. MPT was tested against Echinococcus granulosus protoscolices in vitro. The result showed that MPT could be metabolized in the three biological media, and to the active compound fenbendazole in liver homogenate, with a metabolic rate of 7.92%. Besides, the prodrug showed a weak activity against E. granulosus protoscolices with a mortality of 45.9%.

Survey on Human Soil-borne Nematode Infection in Xining City

ZHAO Sheng-hu1, HAN Xiu-min2, LEI Wen2

2013, 31(1):  18-71-72. 

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Five fields were selected from Xining City by stratified cluster sampling method for the survey. 4 589 people above 3 years old were examined for nematode infections using Kato-Katz method and children under 12 years old were detected for pinworm infection using transparent tape method from June to August in 2011. The results showed that the total nematode infection rate was 3.0% （136/4 589） with the highest of 3.8% （123/3 284） in rural area. The major species was Ascaris lumbricoides, and the infection rate in 15-20 age group was 1.5%  (4/264）, which was significantly lower than that of the age groups of 60-70 （6.9%, 23/335）, above 70 （5.3%, 6/114） and of 10-15 （5.1%, 19/372）（P<0.05）. The prevalence of A. lumbricoides among the preschool children（9.5%, 12/127） was statistically higher than those in other occupation groups （P<0.05）, and the infection rate showed no statistical significance by gender, ethnic and degree of education （P>0.05）. Pinworm infection in children under 12 years old was only 0.5% （2/437）.

Secretory Expression of Salivary ATP Diphosphohydrolase(Apyrase)from Aedes albopictus in Pichia pastoris

WU Song-quan1 *，WANG Guang-li1，LEI Yong-liang2，ZHANG Kai-bo1

2013, 31(1):  19-73-75. 

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The gene-coding mature apyrase protein from Aedes albopictus was amplified by RT-PCR and cloned in frame with the α-factor secretion signal peptide into Pichia pastoris secreting expression vector pGAPZα-A resulting in the pGAPZα-A-apyrase. After being linearized by BlnⅠ restriction enzyme， the recombinant pGAPZα-A-apyrase was trans-formed into Pichia pastoris GS115 by electroporation. Recombinant strains pGAPZα-A-apyrase/GS115 were screened on YPDS plates containing Zeocin and identified by PCR. The recombinant protein of apyrase (Mr 60 000) has been expressed in the supernatant of Pichia pastoris.

Investigation on the Infection of Blastocystis hominis in Populations in Bama Yao Autonomous County of Guangxi

HE Shan-shan1, WU Ling-yuan1, LIU Xiao-quan1, SHI Huan-huan1*, CHEN Zhi2,

2013, 31(1):  20-76-77. 

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497 fecal specimens were collected from 5 randomly selected villages of Bama County in December 2011, and tested for Blastocystis hominis infection using improved centrifugal sedimentation with hydrochloric acid-ether. Data were analyzed by villages, gender, occupation, age groups and ethnic populations. The results showed that 215 people of 497 were positive, with a prevalence of 43.3%（215/497）. Pandang village had the highest infection rate of 55.7%（68/122）, significantly higher than the other villages（P<0.05）. There was no significant difference in genders, occupations, age groups and ethnic populations（P>0.05）.

Laboratory Testing for a Case of Imported Plasmodium ovale Infection in Zhejiang Province

WANG Xiao-guang*, LEI Yong-liang, LAN Jin-quan, MEI Jian-hua, LI Zu-huo

2013, 31(1):  21-78-79. 

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Blood sample obtained from a patient, which returned from Equatorial Guinea,  with clinical diagnosis of Plasmodium infection was confirmed as imported P. ovale infection by etiology and molecular biological methods. 50 μl blood was obtained before taking anti-malarial drugs to make thin and thick blood smears, Giemsa stained, and observed by microscopy. Genomic DNA was extracted from the blood sample, and detected for DNA fragment of P. ovale, P. vivax, P. falciparum or P. malariae by real-time fluorescent quantitative PCR. P. ovale parasites were found in both thin and thick blood smears, and confirmed by quantitative PCR. With the results of laboratory testing, epidemiological history and clinical manifestations, the patient was diagnosed as imported P. ovale infection.