目的获取日本血吸虫（si）精氨酸酶（ARG）新基因的DNA编码序列及其启动子序列。实验验证ARG基因编码序列的完整性。方法以日本血吸虫成虫DNA做模板，PCR扩增ARG基因编码区的DNA序列并测序；根据siARG基因已扩增的DNA序列设计2条巢式引物，用TaKaRaLATaqPCRCloninginritroKit，扩增siARG基因的启动子序列；对已扩增得到的序列进行TATA盒的寻找以分析启动子序列的位置，实验验证我们曾获取的sjARG新基因编码序列的完整性。结果扩增得到长约1OOObpARG基因DNA序列，siARG基因DNA序列内没有内含子，与cDNA序列完全一致。对启动子序列扩增后，得到一略大于250bp的序列，测序后分析发现其中有36bp与ARG基困DNA序列的5'端重叠。因此，将精氨酸酶基因的DNA序列又向前延伸了221bp，与前面得到的序列拼接后得到一条1486bp的总序列，将该序列在NCBI上进行ORF的寻找发现其最长的ORF达1164bp，起始密码子在第156位，终止密码子在第l319位。该起始密码子前32位为TATA盒的位置。因此，确定该SiARG基因全长cDNA编码序列长应为1164bp。结论成功扩增获得了日本血吸虫精氨酸酶基因编码区的DNA序列，其不含有内含子；并扩增得到了该基因的启动子序列，从而获得了siARG基因真正的全长cDNA序列，为进一步的功能鉴定奠定了基础。

目的筛选cDNA文库识别华支睾吸虫新基因，并将所发现的华支睾吸虫溶血磷脂酶（csLysoPLAH）基因编码区克隆到原核表达质粒PGEX-4T-1和真核表达质粒pcDNA3上，为进一步研究其功能奠定基础。方法对华支睾吸虫cDNA文库进行随机筛选并测序，在NCBI和ExPasy网站上进行序列分析，识别华支睾吸虫新基因，并应用Motif-san、NCBIConservedDomainSearch等程序对其进行结构域分析。根据PGEX-4T-1和pcDNA3上的克隆位点及所发现的sLysoPLAHcDNA编码序列设计引物，进行PcR扩增，扩增产物回收纯化后克隆到原核表达载体PGEX-4T-1和真核表达载体pcDNA3上。构建的PGEX-4T-1-csLysoPLAH、pcDNA3-csLysoPLAH重组表达质粒经PcR、双酶切及测序证实。结果发现sLysoPLAH基因，完整阅读框含708个碱基，编码235个氨基酸，理论分子质量为25.2628ku，理论pI为6.03。序列分析表明，csLysoPLAH编码氨基酸序列与其它物种有较高的同源性，csLysoPLAH具有磷脂酶/羧酸酯酶保守功能域和完整的脂酶保守功能域，并含有丝氨酸酯酶特征性的标签肽（即GxsxG）。所构建的重组原核和真核表达质粒经PCR、双酶切及测序证实与目标基因相符。结论发现华支睾吸虫溶血磷脂酶基因，并成功构建原核和真核重组表达质粒。

构建日本血吸虫的翻译控制肿瘤蛋白（SjTCTP）基因的杆状病毒重组供体质粒pFASTA-TCTP，用该重组转移质粒转化含有杆状病毒表达载体以构建BacmidGFP-TCTP，感染昆虫细胞进行表达。方法将已克隆的sjTCTP基因与线性化的pFAslA进行连接为pFAsTA-TCTP，使pFASTA-TCTP与DH10BAC感受菌进行转座重组，利用抗性及蓝白斑筛选重组BacmidGFP-TCTP克隆，提取BacmidGFP-TCTP转染昆虫细胞Sf9获得有感染力的病毒，利用病毒感染sf9细胞进行蛋 白表达。通过荧光镜观察、RT-PCR、SDS-PAGE和western-Blotting检测表达情况。结果获得了重组sjTCTP的杆状病毒，细胞能表达出与sjTCTP单抗结合的、分子量为23kDa左右的融合有组氨酸标签的目的蛋白。结论SjTCTP能在昆虫细胞中获得良好表达，为其的分子生物学活性研究奠定了基础。

目的构建融合基因IFN-alb/CSPII的原核表达裁体并予以表达：方法采用聚合酶链反应（PCR）从人基因组DNA中扩增出IFN-alb基因，克隆入原核表达载体pGEX-4T-1，构建原核表达载体pGEX-4T-1/IFN-alb利用PCR法从恶性疟原虫基因组DNA中扩增出环子孢子蛋白II区（CSPⅡ）基因。克隆入原核表达裁体pGEX-4T-1构建原核表达载体pGEX-4T-1/CSPII用限制性内切酶BamHI和EcoRI将IFN-alb从原核重组质粒pGEX-4T-1/IFN-alb中切下，克隆入经相同酶切的原核重组质粒pGEX-4T-1/CSPII中，构建融合基因的原核表达裁休pGEX-4T-1/IFN-alb/CSPⅡ融合基因IFN-alb/CSPⅡ经异丙基-β-D-硫代半乳糖苷（IPTG）诱导，在大肠埃希茵中进行初步表达：结果构建的原核表达载体pGEX-4T-1/IEN-albpGEX-4T-1/CSPII和pGEX-4T-1/IEN-alb/CSPII经PCR和酶切鉴定与预期结果一致、证实融合基因IFN-alb/CSPⅡ拼接成功并正确地克隆人原核表达载体。在大肠埃希菌中表达出融合蛋白IFN-alb/CSPII。该融合蛋白经十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分忻与理论预测值相符：经蛋白质印迹法（Westernblotting）鉴定具有免疫原性：结论构建了融合基因IFN-alb/CSPII的原核表达裁体，并在大肠埃希菌中表达了融合蛋白IFN-alb/CSPⅡ。

目的扩增日本血吸虫（Si）p50亲免素（IP）基因全长eDNA和DNA序列，进行原核克隆和研究其免疫学特性。方法从sj成虫RNA中RT。PCR扩增SjpSO亲免素基因完整的编码阅读框。从sj成虫基因组中扩增其DNA序列，对其eDNA序列和DNA序列进行比较分析。寻找内含子。将其eDNA序列克隆人pET30a（+）体中，原核表达并纯化表达产物，Western。blot比较重组蛋白与sj体内相应天然蛋白的免疫原性和免疫反应性，间接免疫荧光法对p50亲免素进行组织定位。结果Sjp50IP基因DNA序列与eDNA相比，有6个内含子；重组p50IP与sj体内的天然蛋白具有相同的免疫学特性，该蛋白位于sj成虫的被膜上。结论成功扩增得到了Sjp50IP基因的全长编码区的eDNA序列和DNA序列，其DNA序列中有6个内含子：重组Sjp50IP的免疫学特性支持将该基因作为疫苗进行进一步的研究。

为识别和克隆华支睾吸虫新基因，对华支睾cDNA质粒文库进行随机筛选并测序，并利用在线生物信息学工具进行序列分析，识别华支睾吸虫未知基因，同时根据PGEX-4T-1多克隆位点及未知基因的序列设计引物，PCR扩增目的基因，并构建原核重组质粒。结果发现了CsvpB基因，其完整阅读框含762个碱基，编码254个氨基酸，理论分子量为27.7kDa。序列分析表明，CsvpB蛋白与其它物种的卵黄前体蛋白有较高的同源性，所构建的重组原核表达质粒PGEX-4T-1-vpB经PcR、双酶切及测序证实与目标基因相符。

From a Clonorchis sinensis adult worm cDNA library, we isolated a cDNA clone encoding a novel lactate dehydrogenase (LDH) gene which encoded a putative protein with a predicted molecular weight of 35.6 kDa. The optimum pH and temperature for the enzyme were 7.5 and 50℃ in the pyruvate reduction while 11 and 80℃ in the lactate oxidation reaction, respectively. CsLDH showed no substrate inhibition by high lactate and NAD+ concentration, and the optimal pyruvate and optimal NADH concentrations were 10 and 0.5mmol/l, respectively. The relative activities of these 2-oxocarboxylic acids were pyruvic acid>2-ketobutyrate> oxalacetic acid>α-ketoglutaric acid>phenylpyruvate. The cofactor 3-acetylpyridine adenine dinucleotide was much more effective than NAD+. The cofactor analogs in which the nicotinamide ring is replaced by 3-pyridinealdehyde were lower activity cofactors, while the nicotinamide ring is replaced by nicotinic acid or thionicotinamide which is not a cofactor to CsLDH. The succinic acid and malic acid are not substrates of CsLDH. Cu2+, Fe2+, and Zn2+ greatly inhibited the CsLDH activity both in the direction of pyruvate reduction and in the direction of lactate oxidation. The inhibition of CsLDH by gossypol may make gossypol a potential therapy drug or a lead compound for C. sinensis. Accordingly, the CsLDH may be a novel potential drug target.

Objective To amplify the hexokinase (HK) gene of Plasmodium falciparum isolate FCC1/HN, compare the sequence with that of other P. falciparum isolates and human, and construct its prokaryotic and eukaryotic expres-sion plasmlds. Methods The HK gene was amplified by PCR from the genomic DNA of P. falciparum isolate FCC1/HN. The amplified HK gene was cloned into the prokaryotic and eukaryotic expression vectors, pET-30a(+) and pcD-NA3, and transferred into Escherichia coli BL21/DE3 and JM109, respectively. The positive recombinant pET-30a(+)-HK and pcDNAa-HK were screened and identified by endonuclease digestion and PCR. The nucleotide sequence of HK gene was determined by the dideoxy chain termination method. The DNA sequence and its deduced protein sequence were analyzed. Results The HK gene of P. falciparum isolate FCCI/HN was specifically amplified, and the correct recom- binant plasmids pET-30a(+)-HK and pcDNA3-HK were constructed. The results of sequencing the nucleotide acids showed that the full length HK gene of P. falciparum isolate FCC1/HN was 1 482 base pair. It encoded a protein of 493 amino acids. The amino acids sequence was the same as that of isolate 3D7, with 99.8% identity of the isolate K1, but with 23.2%-26.6% identity of all the 4 types of the human HK. Conclusion The HK gene of P, falciparum isolate FCCI/HN was amplified. Its recombinant prokaryotic and eukaryotic expression plasmids were successfully constructed and its nucleotide sequence were determined. The HK gene of P. falciparum is quite conserved among different isolates, but has a low homology with human HK. The HK of P. falciparum may be a potential drug target.

Some effective antiparasitic drugs for chemotherapy schistosomiasis, such as praziquantel and artemether, are proved to act on lactate dehydrogenase (LDH) in vitro, but detailed molecular action mechanisms of these have not yet been elucidated. To this end, the sequence encoding LDH of Schistosoma japonicum (SjLDH) was amplified from the cDNA library of the adult parasite. Sequence analysis revealed an open reading frame (ORF) of 999 bp, encoding 332 amino acids with a molecular weight of 36.120 kDa. It has three transmembrane regions and may be located in the cytoplasm membrane. The encoding ORF was inserted into pET-28a vector and expressed in Escherichia coli (E. coli) induced by IPTG. A 36-kDa protein with 6

目的了解编码SAG3的重组质粒和IL-2基因佐剂在小鼠体内的免疫反应，为进一步的核酸疫苗及免疫学研究创造条件。方法将编码SAG3的质粒和鼠源性IL-2表达载体以100μg的剂量感染小鼠，3周中两次以相同的剂量加强免疫，分别以PBs和空质粒pcDNA3、1感染。采用ELISA法测定抗体水平、亚型、IFN-γ、IL-4和IL-2的含量，RT-PCR方法检测注射部位目的基因的转录，所有鼠均由强毒力ZS2株弓形虫感染。结果sAG3表达质粒3次免疫后鼠体内特异IgG水平明显上升，IL-2表达质粒的联合使用导致IgG2a水平的升高和IFN-γ的产生，提高了其分泌IL-2的水平，但对IL-4的水平产生轻微的影响；RT-PcR显示首次使用导致IgG2a水平的升高和IFN-γ的产生，提高了其分泌IL-2的水平，但对IL-4的水平产生轻微的影响；RT-PCR显示首次免疫15天后，目的基因在肌肉组织中仍有表达；混合质粒注射和小鼠抗弓形虫感染的存活时间延长。结论由SAG3DNA诱导的免疫应答因IL-2表达质粒的共同注射而增强，DNA疫苗和适当细胞因子的共注射对抵抗弓形虫感染的效果值得进一步研究。