Research progress of copy number variations associated with infantile spasms / 中华实用儿科临床杂志

Infantile spasms (ISs) is an age-dependent epileptic encephalopathy, which is highly associated with cognitive impairment, autism spectrum disorder and developmental delay.At present, inheritance patterns, including chromosomal abnormalities, copy number variations (CNVs), and monogenic diseases have been found to correlated with the etiology of ISs.Among them, CNVs or structural chromosome rearrangements are of great significance in the pathogenesis of ISs.This article aims to review the research progress of CNVs and ISs, thus providing references for further clarifying the molecular mechanism and genetic basis of ISs.

Prognostic value of gastroepiploic lymph node metastasis in transverse colon cancer / 中华消化外科杂志

Objective:To investigate the prognostic value of gastroepiploic lymph node (GLN) metastasis in transverse colon cancer.Methods:The propensity score matching and retrospective cohort study was conducted. The clinicopathological data of 371 patients with transverse colon cancer who were admitted to Fujian Medical University Union Hospital from November 2010 to November 2017 were collected. There were 202 males and 169 females, aged from 21 to 92 years, with a median age of 58 years. Patients were performed complete mesocolic excision combined with GLN dissection by one group of surgeons. Of the 371 patients with transverse colon cancer, 15 cases had positive GLN metastasis (GLN+), and 356 cases had negative GLN metastasis (GLN-). Observation indicators: (1) the propensity score matching conditions and comparison of baseline data between GLN- patients and GLN+patients with transverse colon cancer after propensity score matching; (2) follow-up and survival of GLN- patients and GLN+patients with transverse colon cancer; (3) influencing factors for prognosis of patients with transverse colon cancer. Patients were followed up by outpatient examination or telephone interview to detect tumor metastasis and survival. Follow-up was conducted once every 3 months within postoperative 2 years, once every 6 months within postoperative 2-5 years and once a year thereafter up to January 2020. The propensity score matching was conducted by 1∶4 matching using the nearest neighbor method. Measurement data with skewed distribution were described as M (range), and comparison between groups was analyzed using the rank sum test. Count data were represented as absolute numbers, and comparison between groups was analyzed using the chi-square test or Fisher exact probability. The Kaplan-Meier method was used to calculate survival rates and draw survival curves, and Log-rank test was used for survival analysis. Univariate and multivariate analyses were performed using the COX proportional hazard regression model. The variables with P<0.10 in the univariate analysis were included for multivariate analysis. Results:(1) The propensity score matching conditions and comparison of baseline data between GLN- patients and GLN+ patients with transverse colon cancer after propensity score matching: 55 of 371 patients had successful matching, including 44 GLN- patients and 11 GLN+ patients. Before propensity score matching, the age, cases in stage 0 or stage 1 of M staging, preoperative carcinoembryonic antigen were 60 years(range, 24-92 years), 328, 22, 4.1 μg/L(range, 0.2-343.7 μg/L) for GLN- patients, respectively, versus 67 years(range, 21-79 years), 11, 4, 5.0 μg/L(range, 0.7-952.4 μg/L) for GLN+ patients, showing significant differences in the above indicators between the two groups ( Z=-1.440, χ2=9.031, Z=-2.086, P<0.05). After propensity score matching, the above indicators were 58 years(range, 45-67 years), 40, 4, 4.0 μg/L(range, 2.0-10.0 μg/L) for GLN- patients, respectively, versus 67 years(range, 59-71 years), 9, 2, 5.0 μg/L(range, 8.0-19.0 μg/L) for GLN+ patients, showing no significant difference between the two groups ( Z=-1.580, χ2=0.105, Z=-0.821, P>0.05). (2) Follow-up and survival of GLN- patients and GLN+ patients with transverse colon cancer: GLN- patients and GLN+ patients with transverse colon cancer were followed-up for 12-92 months and 1-70 months, with a median time of 53 months and 30 months respectively. Three cases of GLN- patients and 2 cases of GLN+patients had postoperative liver metastasis, respectively, showing no significant difference between the two groups ( χ2 =0.344, P>0.05). One case of GLN- patients and 3 cases of GLN+ patients had heterochronous lung metastasis, respectively, showing a significant difference between the two groups ( χ2 =4.870, P<0.05). The 5-year disease progression-free survival rates were 82.3% and 33.9% for GLN- patients and GLN+ patients, respectively, showing a significant difference between the two groups ( χ2 =13.366, P<0.05). (3) Influencing factors for prognosis of patients with transverse colon cancer: results of univariate analysis showed that pT staging, pN staging, M staging and GLN metastasis were related factors for prognosis of patients with transverse colon cancer ( hazard ratio=1.599, 5.107, 4.511, 6.273, 95% confidence interval as 0.467-5.471, 1.867-13.971, 1.385-14.694, 2.052-19.176, P<0.05). Results of multivariate analysis showed that pN staging, M staging and GLN metastasis were independent influencing factors for prognosis of patients with transverse colon cancer ( hazard ratio=6.399, 6.163, 4.024, 95% confidence interval as 2.028-20.189, 1.666-22.800, 1.177-13.752, P<0.05). Conclusion:For the patients with transverse colon cancer, GLN metastasis is associated with high postoperative heterochronous lung metastasis rate and poor prognosis. GLN metastasis is an independent prognostic factor for patients with transverse colon cancer.

Effect of all-trans-retinoic acid on proliferation of human lung adenocarcinoma cell line A549 and expression of APLNR gene / 临床检验杂志

Objective To explore the effects of all-trans-retinoic acid (ATRA) on the proliferation of human lung adenocarcinoma cell line A549 and the expression of APLNR (apelin receptor) gene.Methods The inhibition of proliferation of human lung adenocarcinoma cell lines A549 cultured in vitro with or without ATRA was measured by MTT (methyl thiazolyl tetrazolium,MTT) method.The morphological changes in the cells were observed by light microscopy.The cell cycle and apoptosis were analyzed by flow cytometry.The levels of APLNR,cyclin D1 and p16 proteins were detected by western blot.Results After treatment of ATRA,the proliferation of A549 cells was obviously inhibited in dose-and time-independent manner (P ＜ 0.01).The cell morphology was significantly changed.The cycle of A549 cells was blocked at G0/G1 phase and the apoptosis rate was increased.With the increasing concentration of ATRA,the expressions of cyclin D1 and APLNR were down-regulated but the expression of p16 was up-regulated (P ＜ 0.01).Conclusion ATRA could inhibit the proliferation of A549 cells by retardant cell cycle of A549 cells at G0/G1 phase and inducing the apoptosis,and down-regulate the expression of APLNR gene.

Observation on destroyed architecture of splenic lymphoid follicles in mice infected with Schistosoma japonicum by immunohistochemistry / 中国血吸虫病防治杂志

Objective To observe the destroyed architecture of splenic lymphoid follicles in mice infected with Schistosoma japonicum by immunohistochemistry. Methods The mice infected with S. japonicum(20 cercariae/mouse)for 8 weeks were sacrificed,and the splenic samples were paraffin embedded and sliced. The sections were first stained by hematoxylin and eosin to observe the massive structure of splenic lymphoid follicles,and then B cells,follicular dendritic cells(FDC)and germinal center cells were labeled with anti-B220,anti-CD21 or anti-Ki67 antibodies respectively by immunohistochemistry to observe the distribution of the specific cells of lymphoid follicles. Results The results of HE staining showed that the structure of lym-phoid follicles in spleens of infected mice was blurred,the number and area of follicles were significantly reduced compared to those of the normal mice. The immunohistochemical staining showed that the splenic T/B lymphocyte segregation ,FDC network and germinal centers of the infected mice all disappeared. Conclusion The structure of splenic lymphoid follicles in the mice infected with S. japonicum is obviously damaged.

Isolation and identification of macrophages from liver of Microtus fortis / 中国血吸虫病防治杂志

Objective To separate and purify intrahepatic macrophages from Microtus fortis Mf and identify its phagocy?tosis. Methods The intrahepatic macrophages from Mf were separated and purified by perfusion collagenase digestion and density gradient centrifugation. The function of the cells was identified by FACS analysis and ink phagocytosis activity. Results The macrophage cells from the liver of Mf were obtained. These cells were bright and circular and grew adhering to the wall. The proportion of the living cells was 95%. The binding rate of these cells from Mf with anti?mouse CD14 antibody Clone Sa2?8 was about 50%of the rate of macrophage from C57BL/6 mice with this monoclonal antibody. The result of ink?phagocytosis ex?periment of macrophage cells from the liver of Mf was positive. Conclusion The method above mentioned is useful to separate and purify macrophage from the liver of Mf. The study builds the foundation for further research on macrophages of Mf against Schistosoma japonicum.

Molecular characteristics and RNA interference efficiency of Schistosoma ja-ponicum Sj79 gene / 中国血吸虫病防治杂志

Objective To study the structural features and characteristics of a novel gene Schistosoma japonicum 79(Sj79), and observe its effect of RNA interference(RNAi),so as to provide the experimental basis for its further function study and mechanism study of anti reproductive development of schistosome. Methods The gene structure and characteristics of Sj79 were analyzed by bioinformatics methods. Then the expressions of Sj79 messenger RNA(mRNA)during the different develop?mental stages of schistosome were analyzed and the effects of RNAi silencing were observed by the soaking method. The tran?scriptional levels of Sj79 after RNAi were detected by real time PCR. Results The open reading frame of Sj79 contained 696 base pairs with an exon structure. The gene had obvious stage specificity,and its transcriptional level in mature female worms was the highest. After soaking for 3 d,the Sj79 mRNA level[(41.0 ± 12.3)%]in the siRNA?1 group with low dosage(20 nmol/L) was lower than that in the siRNA?NC group[(103.2 ± 14.4)%],the difference was statistically significant(t=3.28,P<0.05). When with high dosage(200 nmol/L ),both the Sj79 mRNA levels in the siRNA?1 group[(15.8 ± 10.9)%]and siRNA?2 group [(11.1 ± 8.8)%]were significantly lower than that in the siRNA?NC group[(100.1 ± 6.3)%](t=13.44,27.84,both P<0.01). After soaking for 7 d,only the Sj79 mRNA levels in the siRNA?1group[(43.4 ± 4.5)%]and siRNA?2 group[(62.5 ± 5.4)%]with low dosage were lower than that in the siRNA?NC group[(100.4 ± 5.2)%],and the differences had statistical sig?nificance(t=8.33,5.07,both P<0.01). Conclusion Through this study,we have improved the mRNA sequence and genom?ic information of Sj79 gene,and understood its structural features,as well as selected out two effect fragments siRNA?1and siR? NA?2 which will provide the basic evidences for the further study on egg laying interference of the female adult worm of schisto?some in vitro.

Relationship between vascular disease and uric acid of type 2 diabetic patients / 中国综合临床

Objective To investigate the correlation between uric acid level and macrovascular disease in type 2 diabetic patients.Methods Sixty type 2 diabetic patients with lower limb atherosclerosis of carotid artery were randomly selected in study group who hospitalized in the First Hospital of Zibo from Mar.to Feb.2012.Sixty type 2 diabetes mellitus(T2DM) without carotid and lower limb athemsclerosis were served as control group.The blood pressure,blood lipid,blood glucose and other biochemical indexes,including blood uric acid,serum insulin (FNS),fasting blood glucose (FPG),apolipoprotein a (LP (a)),apolipoprotein A1,B (APO-A1,APO-B),glycosylated hemoglobin (HbA1c),high density lipoprotein cholesterol (HDL-C),low density lipopmtein cholesterol (LDL-C),triacylglycerol (TG) and total cholesterol (TC) were measured and determined.Results There was no significant difference in terms of blood pressure,blood lipid levels,APO-A1,APO-B,HbA1C,FNS and FPG in study group patiems (P ＞ 0.05).The level LP(a) in study group was (0.4 ± 0.2) g/L,significantly higher than that in control group ((0.2 ± 0.2) g/L; t =3.842,P ＜ 0.01).The blood uric acid level in study group was (362.3 ± 112.8)mmol/L,significantly higher than that of the control group((284.8 ±68.6)mmol/L;t =3.188,P＜0.01).Conclusion Uric acid and LP(a) are involved in the oocurrence and development of athemsclemsis,which is close related to the development of type 2 diabetic macmangiopathy.Therefore,in the process of preventing type 2 diabetes with macroangiopathy,we should pay attention to uric acid and LP (a) of the patient beside effective control of blood glucose,blood pressure,blood lipid level.

The detection of vascular endothelial growth factor and Periostin in the early diagnosis of diabetic retinopathy / 中国医师进修杂志

Objective To detect the expressions of vascular endothelial growth factor(VEGF) and Periostin in patients with diabetic retinopathy (DR),and to explore its chnical significance.Methods A total of 52 patients with DR (DR group),36 non-DR diabetic patients (non-DR group) and 30 healthy person (normal control group) were enrolled.The 52 cases of DR patients were divided into non-proliferative DR (PDR) group (non-PDR group,24 cases) and PDR group (28 cases).The expressions of serum VEGF and Periostin were detected with enzyme-linked immunosorbent assay analysis.Results The serum expressions of VEGF and Periostin were significantly higher in non-DR group and DR group than those in normal control group [(122.63 ±28.74),(163.58 ±42.37) mg/L vs.(91.53 ± 19.58) mg/L,(110.15 ±32.62),(146.51 ± 41.74) mg/L vs.(82.26 ± 21.17) mg/L,P ＜ 0.05 or ＜ 0.01].The serum expressions of VEGF and Periostin were significantly higher in DR group than those in non-DR group (P ＜0.05).The serum expressions of VEGF and Periostin were significantly higher in PDR group than those in non-PDR group [(174.15 ±47.31) mg/L vs.(147.66 ±38.25) mag/L,(160.31 ±46.43) mg/L vs.(132.14 ±35.62)mg/L,P ＜ 0.05].The serum expression of VEGF was positively related with Periostin (r =0.415,P ＜ 0.01).Conclusion The high-expressions of VEGF and Periostin are found in DR patients,and which maybe play a key role in the early diagnosis and assessment the progression of DR patients.

The identification of the Cryptosporidium ubiquitum in pre-weaned Ovines from Aba Tibetan and Qiang autonomous prefecture in China / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>Cryptosporidium spp. are prevalent globally and sheep are an important zoonotic reservoir. Little data regarding the rates of Cryptosporidium infections in ovines in China are available. This study assessed the prevalence of Cryptosporidium spp. in pre-weaned ovines from Aba Tibetan and Qiang Autonomous Prefecture in the Sichuan province of China.</p><p><b>METHODS</b>A total of 213 fecal samples were collected from pre-weaned ovines and were examined microscopically (following modified acid fast staining). In addition, 18S rRNA genetic sequences were amplified from fecal samples by nested PCR and phylogenetically analyzed.</p><p><b>RESULTS</b>The prevalence of Cryptosporidium in the collected samples was at 14.6% (31/213) and four isolates identified by PCR belonged to the Cryptosporidium cervine genotype (Cryptosporidium ubiquitum) demonstrating that this species was the primary sheep species found in sheep in China.</p><p><b>CONCLUSION</b>The present study suggested that the high incidence of Cryptosporidium in sheep poses a significant public health threat and that surveillance practices must be established to prevent zoonotic disease of humans.</p>

Stroke-associated Infection:382 Serial Case Study / 中国康复理论与实践

@#Objective To study the incidence,risk factors and prognosis of stroke-associated infection(SAI) in acute stroke patients.Methods 382 serial acute cerebral stroke patients were retrospectively surveyed.Results The incidence of SAI was 29.3% in all cases,mainly involved lower respiratory tract(63.0%) and urinary tract(28.4%).Escherichia coli,Coagulase negative staph and Streptococcus viridans were the main pathogenic organisms.Developments of SAI were closely related with aging,diabetes mellitus,the application of antimicrobial agents and invasive procedure.The mortality was 10.7%(12/112) in the cases with SAI,but 1.9%(5/270) in the cases without SAI.Conclusion SAI is caused by various factors in acute stroke patients.Controlling SAI can help to succeed in treating stroke.

Effect of Ultrasonic Therapy on Neurological Function after Cerebral Infarction / 中国康复理论与实践

@#Objective To investigate the effect of the ultrasonic therapy on the neurological function after cerebral infarction. Methods 223 cases with cerebral infarction were divided into two groups: treatment group and control group. Both groups received the similar treatment with drug, while the patients in treatment group were treated with the ultrasonic therapy. They were assessed with Clinical Neurological Functional Deficit before and after treatment. Results The scores of Neurological Functional Deficit of all patients decreased (P<0.05), and treatment group decreased more significantly than control group (P<0.05). The total effective rate in the treatment group was higher than that in control group (P<0.05). Conclusion The ultrasonic therapy is helpful for the functional recovery of the patients after cerebral infarction.

Effect of Ultrasonic Therapy on Neurological Function after Cerebral Infarction / 中国康复理论与实践

@#Objective To investigate the effect of the ultrasonic therapy on the neurological function after cerebral infarction. Methods 223 cases with cerebral infarction were divided into two groups: treatment group and control group. Both groups received the similar treatment with drug, while the patients in treatment group were treated with the ultrasonic therapy. They were assessed with Clinical Neurological Functional Deficit before and after treatment. Results The scores of Neurological Functional Deficit of all patients decreased (P<0.05), and treatment group decreased more significantly than control group (P<0.05). The total effective rate in the treatment group was higher than that in control group (P<0.05). Conclusion The ultrasonic therapy is helpful for the functional recovery of the patients after cerebral infarction.

Effect of Ultrasonic Therapy on Neurological Function after Cerebral Infarction / 中国康复理论与实践

@#Objective To investigate the effect of the ultrasonic therapy on the neurological function after cerebral infarction. Methods 223 cases with cerebral infarction were divided into two groups: treatment group and control group. Both groups received the similar treatment with drug, while the patients in treatment group were treated with the ultrasonic therapy. They were assessed with Clinical Neurological Functional Deficit before and after treatment. Results The scores of Neurological Functional Deficit of all patients decreased (P<0.05), and treatment group decreased more significantly than control group (P<0.05). The total effective rate in the treatment group was higher than that in control group (P<0.05). Conclusion The ultrasonic therapy is helpful for the functional recovery of the patients after cerebral infarction.

Effect of Ultrasonic Therapy on Neurological Function after Cerebral Infarction / 中国康复理论与实践

@#Objective To investigate the effect of the ultrasonic therapy on the neurological function after cerebral infarction. Methods 223 cases with cerebral infarction were divided into two groups: treatment group and control group. Both groups received the similar treatment with drug, while the patients in treatment group were treated with the ultrasonic therapy. They were assessed with Clinical Neurological Functional Deficit before and after treatment. Results The scores of Neurological Functional Deficit of all patients decreased (P<0.05), and treatment group decreased more significantly than control group (P<0.05). The total effective rate in the treatment group was higher than that in control group (P<0.05). Conclusion The ultrasonic therapy is helpful for the functional recovery of the patients after cerebral infarction.

Cloning of cDNA encoding Schistosoma japonicum tropomyosin and its expression in Escherichia coli / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To perform cloning of the gene encoding Chinese Schistosoma japonicum tropomyosin (SjcTM) and its expression in Escherichia coli.</p><p><b>METHODS</b>SjcTM cDNA fragment, except for 14 amino acids at the amino terminus, was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) with total RNA extracted from adult worms of S. japonicum. The RT-PCR product was cloned into T vector and sequenced. The SjcTM cDNA, derived from the constructed TA clone pGEM-SjcTM, was then subcloned into the expressing vector pBV220. After characterization by agarose gel electrophoresis, endonucleases digestion and PCR, the resultant recombinant plasmid was used for expression under the temperature-dependent condition.</p><p><b>RESULTS</b>The RT-PCR product, cloned into a T vector, was sequenced and shown to be 96.5% identical at the nuclei acid level and 98.1% identical in deduced amino acid sequence to that of S. mansoni tropomyosin. The target DNA fragment was then subcloned into a prokaryotic vector pBV220. Induced expression in E. coli DH5alpha cells resulted in a constant level of recombinant protein production. The results of SDS-PAGE and Western blot revealed that the molecular weight of non-fusion recombinant protein (rSjcTM) was approximately 32 kDa and could be recognized specifically by a polyclonal antiserum specific for native S. japonicum tropomyosin (SjcTM).</p><p><b>CONCLUSION</b>The engineering of the cDNA encoding S. japonicum tropomyosin and its bacterial expression was successfully made.</p>

A Study on the Recombinant 26 kDa Glutathione-S-Transferase as a Vaccine Candidate:Dynamics of Antibodies in Immunized Buffaloes and Protection against Schistosoma japonicum Infections / 中国寄生虫学与寄生虫病杂志

ObjectiveTo observe the dynamics of antibodies and protection against Schistosoma japonicum infections in buffaloes after immunized with recombinant 26 kDa glutathione S transferase (reSjc26GST). Methods Buffaloes in 2 villages endemic for schistosomiasis japonica were selected as test and control groups, respectively.In test group initially 96 buffaloes were vaccinated with reSjc26GST, and 90 buffaloes in the control group did not experience vaccination. The indicators included levels of antibodies to reSjc26GST in buffaloes before and after infection with S japonicum and changes in infection rate. Results Specific antibodies, which showed a trend of trapezoid increase, were induced in buffaloes after immunized with reSjc26GST. Twenty months after immunization, the infection rate of the test group was decreased by 62 2% when compared with that before vaccination,and by 67 7% when compared with that of the control in the corresponding period.Conclusion Specific antibodies and a certain extent of protection were induced in buffaloes after immunized with reSjc26GST, which played an significant role in ameliorating morbidity.

Immunoscreeening of Schistosoma japonicum schistosomula cDNA library with sera from Microtus fortis and bioinformatic analysis of novel genes / 中国血吸虫病防治杂志

Objective To obtain novel vaccine candidate antigens against Schistosoma japonicum. Methods S. japonicum schistosomula cDNA library was screened by using sera of Microtus fortis that was naturally resistant to schistosomiasis. The positive clones were transformated into Escherichia coli BM25.8, E. coli clones containing the plasmid cultured in LB, and then selected for plasmid extraction, the plasmid DNA was digested by EcoRⅠand Hind Ⅲ, and analysed by agarose gel electrophoresis. The positive clones were also sequenced and the data were analysed through the internet Nucleotide BLAST software of NCBI and Expert Protein Analysis system of GeneRunner and HNN. Results Twelve positive clones were obtained after repeatedly immunoscreening the library and their sizes ranged from 300 bp to 1100 bp. Two novel genes (named as Sj-sMf1 and Sj-sMf2) with complete ORF were obtained. The deduced protein of Sj-sMf1 consisted of 93 amino acids while Sj-sMf2 consisted of 61 amino acids. Sj-sMf1 protein predicted containing one cAMP phosphorylation site and Casein kinase C phosphorylation site, respectively. Sj-sMf1 protein predicted containing one Casein kinase C phosphorylation site and two Protein kinase C phosphorylation sites. Conclusion Two novel genes predictably encoding unknown proteins are obtained from immunoscreening of Schistosoma japonicum schistosomula cDNA library by M. fortis sera.

Cloning and expression of gene encoding myophilin-like protein of Schistosoma japonicum and study on the antigenicity of recombinant protein / 中国血吸虫病防治杂志

Objective To clone and express the gene encoding Schistosoma japonicum myophilin-like protein (SjcMLP) and to study the antigenicity of the recombinant protein. Methods The SjcMLP gene was amplified by PCR. The PCR product was cloned into T vector, and then subcloned into expression vector pQE30. The recombinant plasmid of pQE30-SjcMLP was transformed into E.coli M15, and induced with IPTG for expression. The bacterial lysis was conducted by ultrasonication and the supernatant was analysed by SDS-PAGE. The recombinant protein (reSjcMLP)was purified with the Ni-NTA resin, and analysed with SDS-PAGE and Western blot. The titers of sera from C57BL/6 mice immunized subcutaneously with reSjcMLP were detected by ELISA. Results The results of SDS-PAGE and Western blot showed that the molecular weight of expressed fusion protein was around 24.8 kDa and was recognized by the sera from the mice infected with Schistosoma japonicum. The purified protein of reSjcMLP was coated for ELISA test and the IgG titers in the sera from the mice immunized with reSjcMLP were as high as 1∶12 800 reacted with. However, no significant difference was found in worm reduction rates between the immunized mice and control mice. Conclusions The fused recombinant protein of reSjcMLP is successfully ex-pressed and purified. The recombinant protein in this experiment fails to induce significant protection against the challenge infection in C57BL/6 mice.

Cloning and expression of the gene encoding hypoxanthine-guanine phosphoribosyltransferase of Schistosoma japonicum / 中国血吸虫病防治杂志

Objective To perform the cloning of the gene encoding Schistosoma japonicum Chinese-strain hypoxanthine-guanine phosphoribosyltransferase(HGPRT)and its expression in Escherichia coli.MethodsA couple of primers were designed with the BamHI restriction endonuclease site introduced in forward primer and SalI in reverse primer.Total RNA was isolated from adult worms of S.japonicum Chinese-strain(Anhui-strain,Sjc-A)and the SjcHGPRT gene was amplified by reverse transcriptase-polymerase chain reaction(RT-PCR).The PCR product and the prokaryotic expression vector pET28a were digested by both restriction endonucleases BamHI and SalI.The target DNA fragments were purified and cloned properly into pET28a.After identification by en-donucleases digestion,PCR and sequencing,the recombinant plasmid pET28a-SjcHG PRT was transformed into competent E.coli BL21 and expressed in the presence of IPTG.Results pET28a-SjHGPRT was sequenced and shown to be 99% and 83% identical in deduced amino acid sequence to that of S.japonicum Chinese-strain(Hunan-strain,Sjc-H)and S.mansoni HGPRT,respectively.The results of SDS-PAGE and Western blot revealed that the molecular weight of expressed protein was around 30 kDa and could be recognized by anti-His-G-HPR antibody and sera from mice and human with schistosomasis japonica.Conclusion The recombinant plasmid containing SjcHGPRT cDNA is successfully constructed and its expression protein(reSjcHGPRT)is also successfully purified.

Cloning, Expression and Analysis of the Heat Shock Protein of Cryptosporidium andersoni / 中国寄生虫学与寄生虫病杂志

Objective To clone and express the partial encoding sequence of Mr 70 000 heat shock protein of Cryptosporidium andersoni (CaHSP70) in Escherichia coli and identify the recombinant protein. Methods Total RNA was extracted from oocysts of C.andersoni isolated from Xuzhou, Jiangsu (XZ-BOV). The CaHSP70 gene was amplified by RT-PCR. The PCR product was cloned and then subcloned into pET28a vector, and the recombinant plasmids were transformed into E.coli BL21(DE3) subsequently. The expressed protein induced by IPTG was purified and identified by SDS-PAGE and Western blotting, and was further analyzed by relevant bioinformatics softwares. The specific IgG antibodies in mice immunized by rCaHSP70 were detected by Western blotting and ELISA respectively. Results The deduced amino acid sequence showed to be identical with that of C. andersoni Mr 70 000 heat shock protein (HSP70). The recombinant protein expressed in the form of inclusion body was about Mr 43 000. It could be recognized by anti-His G labeled HRP antibodies and all the sera from mice infected with C. andersoni and children infected with C. parvum as well as sera from mice immunized with rCaHSP70 respectively. The rCaHSP70 possibly had multiple domains and potential antigenic determinants. Phylogenetic analysis showed that XZ-BOV and C. andersoni were in the same clade. ELISA showed that the level of specific antibodies against rCaHSP70 in immunized BALB/c and C57BL/6 mice was significantly higher than that of mice before immunization. Conclusion The recombinant plasmid pET28a-CaHSP70 has been constructed. The purified rCaHSP70 exhibits high antigenicity and seems a potential candidate antigen for immunodiagnosis of cryptosporidiosis.