Novel drug designing rationale against Brugia malayi microfilariae using herbal extracts

Objective:To explore the effect of herbal polyphenolics on filariasis in vitro.Methods:Two herbal extracts,methanolic extracts of roots of Vitex negundo Linn.（Nirgundi） and leaves of Aegle marmelos Juss.（Beal） in different concentrations ranging from 40-80 ng/mL were tested for their antifilarial activity either alone or in combination with diethyl carbonate（DEC）（300μg/mL） and/or H2O2（0.5 mM）.Results:Combination of DEC and each extract had significant anti-filarial effect.And fractions of both extracts were not effective as crude herbal extract.Conclusions: Such unique pharmacodynamics reported in this study might provide new drug development stratagem against filariasis.

Inflammatory mediator release by Brugia malayi from macrophages of susceptible host Mastomys coucha and THP-1 and RAW 264.7 cell lines

Objective:To investigate which life stage of the parasite has the ability to stimulate release of pro- or anti-inflammatory mediators from macrophages.Methods:The human macrophage/ monocyte cell line THP-1,the mouse macrophage cell line RAW 264.7 and naive peritoneal macrophages（PM） from the rodent host Mastomys coucha（M.coucha） were incubated at 37℃in 5%CO2 atmosphere with extracts of microfilariae（Mf）,third stage infective larvae（L3） and adult worms（Ad） of Brugia malayi.After 48 hr post exposure,IL-1β,IL-6,TNF-α,IL-10 and nitric oxide（NO） in cell-free supernatants were estimated.Results:Extracts of all the life stages of the parasite were capable of stimulating pro-（IL-1β,IL-6 and TNF-α） and anti-inflammatory （IL-10） cytokines in both the cell lines and peritoneal macrophages of M.coucha.Mf was the strongest stimulator of pro-inflammatory cytokines followed by L3 and Ad;however,Ad was a strong stimulator of IL-10 release.Mf was found to have potential to modulate LPS-induced NO release in RAW cells.Ad-induced NO release was concentration dependent with maximum at 20μg/mL in both RAW and PMs.Conclusions:The results show that parasites at all life stages were capable of stimulating pro-（IL-1β,IL-6 and TNF-α） and anti-inflammatory（IL-10） cytokines and NO release from macrophages of susceptible host M.coucha,human and mouse macrophage cell lines.Mf can suppress the LPS-induced NO release in RAW cells.The findings also show that the two cell lines may provide a convenient in vitro system for assaying parasite-induced inflammatory mediator release.

Immunostimulatory role of rBmHSP60 from filarial parasite Brugia malayi

Objective:To evaluate the immunostimulatory potential of crossreactive molecule heat shock protein 60(HSP60)of filarial parasite Brugia malayi and Leishmania donovani.Methods:HSP60 of Brugia malayi(BmHSP60)was amplified using gene-specific primer,cloned in p Tri Ex4 vector,expressed in BL21-DE3 cells,and recombinant HSP60(rHSP60)of~65 k Da was purified by affinity chromatography using Ni-NTA column.The recombinant protein was desalted by the dialysis membrane,and the presence of endotoxin level was determined by Limulus amebocyte lysate assay.The recombinant protein was tested for cell proliferation,nitric oxide release,expression of Th1 and Th2 cytokines,and transcription factors(STATs)in vitro using murine macrophage cell line(J774 A.1).Results:Higher cell proliferation indicated that BmHSP60 had immunostimulatory potential.rBmHSP60 exposure upregulated the expression of iNOS,STAT1,STAT4,Th1 cytokines(IFN-γ,TNF-α,IL-12),and nitric oxide release.In addition,no remarkable change was observed in the expression of IL-6,IL-10,and STAT3 in macrophage cell line J774 A.1.The ELISA analysis showed the levels of IFN-γ,TNF-α,and IL-12 were upregulated while IL-10 level was downregulated,revealing that BmHSP60 triggered a Th1 immune response.Conclusions:Our study demonstrates that rBmHSP60 has immunogenic properties which effectively enhances the Th1 type immune responses,and can be used as an immunoprophylactic agent against leishmaniasis.Furthermore,in vivo studies are in progress to determine the protective role of rBmHSP60 against Leishmania donovani infection in a mouse model.

The Dynamic Study of Specific Antibodies in Sera of Jirds and Mice Experimentally Infected with Brugia malayi

This paper deals with the studies on the changes of serum specific IgM andIgG,and relationship between the antibody level and infected worm stage of Brugiamalayi.The jirds (Meriones unguiculatus)and BALB/cCR mice were infected with threedifferent stages of Brugia malayi by different routes and serum specific IgM and IgGwere detected by ELISA mcthod.The IgM of infected jirds and mice occurred earlierthan IgG,reached peak at 4～8th weeks,and maintained at a certain level during 30weeks.The responses and toles of IgM and IgG might probably be considered as animmunological characferistic of parasitic helminthic infection.The antibody levels inBALB/cCR mice after infection were higher than those in jirds.

Effect of short- and long-term immunization of recombinant disorganized muscle protein-1(rDIM-1) against human filarial parasite Brugia malayi in rodents

Objective: To evaluate the effect of short-term and long-term immunization of recombinant disorganized muscle protein-1(r DIM-1) in rodents against human filarial parasite Brugia malayi.Methods: Recombinant Brugia malayi DIM-1(rDIM-1 bm) protein was cloned, expressed and purified using a Ni-NTA affinity column. Mastomys coucha were immunized with rDIM-1 bm in three immunization schedules: short-term(3-dose of rDIM-1 bm), and long-term(booster doses till 3-and 6-week) and subsequently challenged with infective third-stage larvae of filarial parasite Brugia malayi(L3). Microfilaraemia was monitored in L3 exposed groups on day 90 post larval inoculation(p.l.i.) and continued till day 205 p.l.i. On day 205 p.l.i. all the infected animals were killed and total worm burden was estimated. Cellular proliferative response, macrophage activity, nitric oxide(NO) release, specific IgG and its subtypes, IgE, IgA and Th1(IFN-γ, TNF-ααand IL-2) and Th2(IL-4, IL-5, IL-6, IL-10 and IL-13) cytokine release were determined. Results: Of the 3 different immunization schedules, shortterm immunization(3-dose schedule) showed better reduction in microfilarial burden(36%-63%) in the peripheral circulation, adult worm load(52%), whereas long-term immunization(3-and 6-week schedule) exerted less effect on peripheral microfilariae count(9%-58%), and adult worm burden(9%-12.5%). Short-term immunization resulted in upregulation of cellular proliferation, macrophages activity, NO release, specific IgG, IgG1, IgG2 a, Ig G2 b, IgE and IgA levels and both Th1(IFN-γ, TNF-α and IL-2) and Th2(IL-4, IL-5, IL-6, IL-10 and IL-13) cytokine release whereas long-term immunization(3-and 6-week schedule) exerted less effect on parasite burden and showed mixed immunological responses. None of the rDIM-1 bm administration schedules induced any pathology in lymphoid tissues, or alteration in mast cell number and granularity. Conclusions: The short-term immunization with rDIM-1 bm(3-dose schedule) induces robust immune responses and protects the host from filarial parasite infection.

周期型马来丝虫副肌球蛋白基因真核表达重组质粒构建

提取周期型马来丝虫总RNA。跟据已知的马来丝虫副肌球蛋白(BmPmy)基因序列,设计合成引物,并引入HindⅢ和BamHⅠ酶切位点,应用RT-PCR技术,扩增BmPmy基因片段,克隆至载体pGEM-T中,经PCR和双酶切鉴定后,亚克隆至真核表达质粒pcDNA3.1(+),成功构建了真核表达载体pcDNA3.1(+)-BmPmy,并转染COS-7细胞后进行RT-PCR分析。转染的COS-7细胞高水平表达周期型马来丝虫副肌球蛋白mRNA,结果与预期相符。

马来丝虫3-磷酸甘油醛脱氢酶基因的克隆、序列分析及编码产物B细胞表位预测

根据GenBank中马来丝虫3-磷酸甘油醛脱氢酶基因(BmG3PD基因)序列设计引物,以马来丝虫mRNA为模板,RT-PCR扩增BmG3PD基因,将其克隆入pGEM-T载体,转化大肠埃希菌(E.coli)DH5α,筛选阳性克隆。经EcoRⅠ和XhoⅠ双酶切及PCR鉴定,获得阳性重组质粒pGEM-BmG3PD,经序列分析及同源性比较,以及对其编码产物进行B细胞表位预测,结果表明PCR扩增的特异性条带为1020bp,与预期相符,与GenBank已知基因序列同源性为99%。编码产物B细胞表位预测,氨基酸区域可能在22～36、242～255、303～318和326～336位。

马来丝虫长爪沙鼠动物模型保种衰退期的观察

目的 观察马来丝虫经长爪沙鼠传代的衰退情况。 方法 用马来丝虫微丝蚴感染中华按蚊 ,收集感染期幼虫 (L3) ,通过腹腔接种感染长爪沙鼠 ,连续观察 3 3代长爪沙鼠体内马来丝虫微丝蚴的发育情况。 结果 从 1974年建立的马来丝虫长爪沙鼠动物模型至今 ,通过 3 3代传代 ,发现随着转种代数增加 ,从第 2 8代起长瓜沙鼠的阳性率逐年下降 ,由 2 8代的 80 %下降至 3 2代的 16% ,到 3 3代时阳性率降为 0。 结论 经过较长期的传代 ,马来丝虫幼虫难以在长爪沙鼠体内发育繁殖。

影响马来丝虫对长爪沙鼠感染率因素的观察

目的：探讨影响马来丝虫对长爪沙鼠感染率的因素。方法：用含马来丝虫感染期幼虫（Ｌ３）生理盐水感染长爪沙鼠，并分别加用抗生素、葡萄糖或培养液ＲＰＭＩ１６４０，观察接种鼠的存活率、感染率和感染度。结果：用含Ｌ３生理盐水组鼠存活率为８０．９％，存活鼠感染率为６０．５％，高感染度率为４３．４％。加用抗生素组，鼠存活率为９８．８％，存活鼠感染率为５２．９％，高感染度率为３０．６％。加用葡萄糖或ＲＰＭＩ１６４０组，鼠存活率（分别为９８．０％和９１．２％）、感染率（分别为６８．８％和６７．７％）和高感染度率（分别为５０．０％和５１．６％）均较高。长爪沙鼠感染马来丝虫浙江株（６代），存活鼠感染率和高感染度率均较贵州株（３１代）高。结论：加用抗生素能提高感染鼠存活率，但其感染率和感染度降低。加用葡萄糖和ＲＰＭＩ１６４０，能提高感染鼠存活率、感染率和感染度。长爪沙鼠对浙江株（６代）较贵州株（３１代）易感。

周期型马来丝虫半胱氨酸蛋白酶基因原核和真核表达质粒的构建

目的构建我国流行的周期型马来丝虫半胱氨酸蛋白酶(BmCP)基因原核和真核表达质粒,为进一步研究奠定基础。方法从周期型马来丝虫虫体中提取总RNA,反转录成cDNA,设计合成引物,以cDNA为模板,通过PCR从cDNA中扩增出目的基因,扩增产物经初步鉴定后将其克隆人pMD18-T载体.进行双酶切及PCR扩增鉴定,获得阳性重组质粒,进行测序分析和同源性比较。阳性克隆的质粒亚克隆至真核表达质粒pcDNA3.1(+).转化感受态大肠埃希菌(E.coli)DH5α,筛选阳性克隆,用PCR和双酶切鉴定阳性重组子。结果RT-PCR扩增出1条约1201 bp的特异性条带,重组质粒双酶切和以质粒为模板的PCR结果与预期相符,DNA序列分析与GenBank已知的基因序列同源性为99%。构建了真核重组表达质粒peDNA3.1-BmCP。结论成功构建了周期型BmCP原核和真核重组表达质粒,为进一步研究该基因的功能提供了条件。

宿主对周期型马来丝虫微丝蚴细胞毒作用的体内研究

目的以我国流行的周期型马来丝虫为研究对象，研究宿主体内清除虫体、减轻虫体负荷、控制丝虫感染的免疫效应机制。方法提取纯净的周期型马来丝虫微丝蚴（ｍｆ）装入微孔实验盒，然后植入 ＳＤ大鼠体内，在不同时间观察机体免疫系统对ｍｆ的杀伤作用。结果含ｍｆ的３．０μｍ微孔盒内，迁入的细胞量随植入时间延长而逐渐增多。迁入的细胞种类及百分率，各实验组之间无明显差异。免疫血清组ｍｆ被效应细胞粘附、杀伤率均明显高于正常血清组。免疫血清用抗ＩｇＧ免疫球蛋白处理后，粘附细胞虫体及虫体死亡率都大大下降。 ３．０μｍ微孔盒内大量效应细胞粘附于虫体表面。扫描电镜下，观察到效应细胞粘附的虫体表面出现坏死、剥脱。结论ｍｆ对宿主效应细胞有一定的趋化作用。宿主体内存在着抗体介导的细胞毒作用，参与细胞毒作用的抗体主要是ＩｇＧ。其主要的效应细胞是中性粒细胞、巨噬细胞和嗜酸粒细胞。

马来丝虫肌球蛋白部分编码基因Bm-M55的克隆与真核表达

根据马来丝虫肌球蛋白部分编码基因(Bm-M55)序列设计引物,以其微丝蚴总RNA为模板,反转录PCR扩增目的基因。用TA克隆方法将目的基因克隆至载体pGEM-TEasy中,经PCR和双酶切鉴定并测序后,亚克隆至真核表达质粒pcDNA3.1(+),构建真核表达载体pcDNA3.1(+)/Bm-M55,转染COS-7细胞后进行RT-PCR验证。用十二烷基硫酸钠-聚丙烯酰胺电泳(SDS-PAGE)对获得重组蛋白Bm-M55进行分析和鉴定。RT-PCR鉴定结果显示,转染的COS-7细胞表达了Bm-M55基因,根据克隆的目的基因序列推导的氨基酸序列与GenBank(登录号为AAA27858)中的一致,重组蛋白Bm-M55相对分子质量(Mr)约为55000。

周期型马来丝虫CPI基因真核表达载体的构建和免疫学研究

目的构建周期型马来丝虫半胱氨酸蛋白酶抑制剂(BmCPI)基因真核表达载体pcDNA3.1-BmCPI,并观察其在小鼠体内的免疫应答反应。方法以周期型马来丝虫总RNA为模板,逆转录PCR(RT-PCR)扩增目的基因片段。与pGEM-T Easy克隆载体连接,筛选出阳性克隆,经PCR和双酶切鉴定后,亚克隆至真核表达质粒pcDNA3.1,构建pcDNA3.1-BmCPI表达载体。将48只小鼠随机分为4组,每组12只,分别为健康对照组、空质粒组、pcDNA3.1-BmCPI组和pcDNA3.1-BmCPI/CpG组,分别注射PBS 100μl、空质粒pcDNA3.1 100μg、重组质粒pcDNA3.1-BmCPI100μg和重组质粒pcDNA3.1-BmCPI 100μg+佐剂CpG 30μg,采用左后腿胫前肌注射免疫。每2周1次,共3次,于末次免疫后第4周,用RT-PCR方法检测小鼠注射部位肌肉组织内目的基因转录情况。于末次免疫后第4和第6周,用噻唑蓝(MTT)法检测小鼠T淋巴细胞刺激增殖水平。于末次免疫后第2、4和6周,用ELISA法检测小鼠血清γ干扰素(IFN-γ)和白介素-4(IL-4)水平。结果成功构建了pcDNA3.1-BmCPI真核表达载体,基因片段大小为621 bp。该真核表达载体免疫小鼠后,从小鼠肌肉组织扩增出目的基因。末次免疫后第4和第6周,2个免疫组小鼠淋巴细胞刺激增殖指数均显著高于健康对照组和空质粒组(53.789±1.937、59.735±4.139和61.975±1.029)(均P<0.05),2个免疫组间差异无统计学意义(P>0.05)。于末次免疫后第2、4和6周,pcDNA3.1-BmCPI组和pcDNA3.1-BmCPI/CpG组小鼠血清IFN-γ水平随时间延长逐渐升高,分别为69.544±3.145和106.069±7.518、120.019±5.968和136.229±7.198、149.109±2.700和178.429±1.126,均显著高于健康对照组和空质粒组(28.264±1.129、35.179±1.029和40.110±1.176)(均P<0.05),其中末次免疫后第2和第6周,pcDNA3.1-BmCPI/CpG组IFN-γ水平显著高于pcDNA3.1-BmCPI组(均P<0.05)。于末次免疫后第4和第6周,2个免疫组的IL-4水平均显著高于健康对照组和空质粒组(均P<0.05),2个免疫组间差异均无统计学意义(均P>0.05)。结论 pcDNA3.1-BmCPI真核表达载体能在小鼠体内转录,并可诱导免疫应答。

周期型马来丝虫半胱氨酸蛋白酶基因的扩增、克隆及序列分析

[目的]克隆周期型马来丝虫半胱氨酸蛋白酶(cysteine protease of periodic Brugiamalayi,BmCP)基因到原核载体中,测定其序列,为进一步的研究奠定基础。[方法]从周期型马来丝虫虫体中抽提总RNA,以mRNA为模板,采用RT-PCR法体外扩增出BmCP基因序列,扩增产物经初步鉴定后将其克隆入pMD18-T载体,转化大肠杆菌(E.coli)DH5α,筛选阳性克隆,进行双酶切及PCR扩增鉴定,获得阳性重组质粒pMD18-T-BmCP,经测序验证,并进行同源性比较。[结果]RT-PCR扩增出一条约1201bp大小的特异性条带,重组质粒双酶切和以质粒为模板的PCR结果与预期相符,DNA序列分析与GeneBank已知的基因序列同源性为99%。[结论]成功构建了周期型马来丝虫半胱氨酸蛋白酶重组质粒pMD18-T克隆载体,为进一步研究该基因的功能提供了条件。

周期型马来丝虫HSP70基因原核表达载体的构建及表达产物的分析

目的 在大肠埃希菌宿主系统中表达周期型马来丝虫热休克蛋白70（HSP70）基因，并对表达产物进行SDS—PAGE分析。方法以重组质粒pGEM—T—HSP70为模板，根据报道的HSP70基因序列设计合成一对引物，用PCR法扩增HSP70基因（包括完整开放读码框架片段及其上下游序列共长2198bp），PCR产物定向插入原核表达载体pGEX-4T-3中，构建的重组质粒pGEX-4T-3-HSP70经双酶切鉴定。将重组质粒转化大肠埃希菌BL21（DE3），用IPTG诱导GST．Tag融合蛋白的表达。经SDS—PAGE分析并经凝胶图像分析确定目的蛋白的表达水平。结果重组表达质粒pGEx-4lT-3-HSP70全长约7200bp，经双酶切后应得到长度为4968bp的载体和2198bp的目的基因2个片段，1％琼脂糖凝胶电泳显示结果同预期完全相符。HSP70相对分子质量（Mr）为70×10^3，质粒pGEX-4T-3的融合部分（GST．Tag）表达产物的胁约为26×103，将重组质粒转化BL-21（DE3）菌。经IPTG诱导表达，菌体蛋白的SDS—PAGE图谱显示，诱导组出现特异性蛋白表达条带，其Mr约100×10^3，与预计大小相同。目的蛋白占菌体总蛋白的12％左右。结论 成功地构建了重组原核表达载体pGEX一4T一3-HSP70：周期型马来丝虫HSP70基因可在大肠埃希菌中稳定表达，为制备和纯化表达蛋白以及丝虫病疫苗的进一步研究打下基础。

感染马来丝虫微丝蚴中华按蚊元素的分析

应用原子吸收光谱分析仪对中华按蚊的羽化蚊、吸正常人血后0d、5d、8d、12d、18d及感染马来丝虫微丝蚴后5d、8d、12d、18d的中华按蚊虫体内13种元素含量的变化进行了分析。这13种元素由钾、钠、钙、镁等4种常量元素和铁、锌、镍、铝、钢、铅、铜、锰、铬等9种微量元素组成。结果表明，非感染马来丝虫微丝蚴中华按蚊体内元素的含量随蚊虫的发育而增减不一，减少明显的有锌、铁、镁、铜等，而钙、钾等却有所增加。感染蚊与非感染蚊相比较，多种元素的含量明显减少，于感染第5d，有铁、锌、钾、钙、钠、铝、钢、铅、铜、锰等10种；第8d有除钙、镁以外的11种；第12d有除钙、镁、铜、镍外的9种；第18d有钾、镍、铝、镉、铅、锰、铬等7种元素的含量显示出非常明显的差异。

PCR及PCR-ELISA法检测蚊体内马来丝虫幼虫

目的：建立一种检测蚊体内马来丝虫幼虫的灵敏、快速、特异的方法。方法：选择应用ＰＣＲ及ＰＣＲ－ＥＬＩＳＡ法检测马来丝虫幼虫ＤＮＡ的最佳反应条件，并在该条件下分别以马来丝虫Ⅰ期、Ⅱ期、Ⅲ期幼虫各１条作模板，测定检测的灵敏度及检测实验室人工感染中华按蚊体内的马来丝虫幼虫。结果：ＰＣＲ及ＰＣＲ－ＥＬＩＳＡ法均能检测出１条Ⅰ期幼虫（Ｌ１），而ＰＣＲ的检测下限为１／１０条Ｌ１，ＰＣＲ－ＥＬＩＳＡ检测下限为１／１００条Ｌ１；将分离的感染期幼虫加阴性蚊媒进行粗提、扩增及电泳，结果未见明显的扩增条带，扩增产物作ＥＬＩＳＡ检测，全部为阴性；个体解剖人工感染的中华按蚊１２０只，分别收集１１３只阳性蚊体内的幼丝虫，用两种方法检测，结果均为阳性。结论：初步建立了ＰＣＲ及ＰＣＲ－ＥＬＩＳＡ法检测蚊体内马来丝虫幼虫的方法。

贵州两个不同地区周期型马来丝虫成虫氨基酸的组成研究

应用高效液相色谱对贵州省内的两个不同地区周期型马来丝虫（贵州独山株、贵州荔波株）成虫的１８种氨基酸进行了检测和分析。结果显示贵州独山株与荔波株马来丝虫均检出１７种氨基酸，两株丝虫有１６种相同氨基酸。两株氨基酸的总含量无明显差异（Ｐ＞０．０５）。但独山株有丝氨酸，缺酪氨酸，而荔波株含酪氨酸，缺丝氨酸。

应用DNA探针检测蚊体内丝虫幼虫的研究

本文用人工合成的寡核苷酸探针，经斑点杂交法检测蚊体内马来丝虫幼虫。可测到丝虫和微丝蚴２ｎｇ的ＤＮＡ量，不与其它动物丝虫标本发生交叉反应。将单个蚊直接压于硝酸纤维素膜上进行检测，感染蚊中含有１条感染期幼虫就可出现阳性反应。将一组蚊虫在裂解液中研磨集体检测时，在２０只蚊虫中有１只感染蚊即可检出。显示该探针可用于马来丝虫地区的蚊媒监测。

我国周期型马来丝虫六个省区虫株氨基酸的比较分析

目的 本实验进行丝虫种内分类学研究探讨我国六个省七个不同地区马来丝虫是否存在种内的生物学特性差异。方法 采用ＨＰＬＣ对贵州独山株、贵州荔波株、四川乐山株、湖北谷城株、安徽泾县株、浙江安吉株、福建建阳株马来丝虫成虫的１８种虫体氨基酸含量及种类进行分析比较。结果 七个地区马来丝虫虫体氨基酸的总含量经统计分析无明显的差异（Ｐ＞ ０．０５），碱性氨基酸、酸性氨基酸以及芳香族氨基酸亦无明显的差异（Ｐ＞ ０．０５），但在一些种类的氨基酸含量高低和个别氨基酸的缺如有一定的差异。结论 七个虫株丝虫虽有微小的生物学差异。