热带医学杂志
熱帶醫學雜誌
열대의학잡지
JOURNAL OF TROPICAL MEDICINE
2002年
3期
219-224
,共6页
单志新%余新炳%徐劲%吴忠道%李学荣%卞国武%马长玲%李焱%陈守义%胡旭初
單誌新%餘新炳%徐勁%吳忠道%李學榮%卞國武%馬長玲%李焱%陳守義%鬍旭初
단지신%여신병%서경%오충도%리학영%변국무%마장령%리염%진수의%호욱초
恶性疟原虫%Pf332抗原%分子克隆%表达%免疫%序列分析
噁性瘧原蟲%Pf332抗原%分子剋隆%錶達%免疫%序列分析
악성학원충%Pf332항원%분자극륭%표체%면역%서렬분석
Plasmodium falciparum%antigen Pf332%c loning,molecular%expression%immunization%sequence analysis
目的了解恶性疟原虫FCC1/HN株Pf332抗原(Ag332)的初级结构和潜在的抗原表位.方法根据Pf332基因已知序列,合成9对引物用于从恶性疟原虫FCC1/HN株基因组DNA 中扩增Pf332基因片段.将扩增得到的9个Pf332基因片段插入pMD-18T载体后测序.用DNAstar软件将测定的Pf332基因片段序列拼接出成完整的 Pf332基因序列.分别用SAPS、Tmpred、SingalP 和Blastn程序来分析Ag332的初级结构和序列同源性.将与Pf332基因的9595～10083、10339～10767 和10855～11247位碱基对应的R0 、R1和 R2片段分别插入真核表达载体pcDNA3-S.将pcDNA3-S-R0、pcDNA3 -S-R1 和pcDNA3-S-R2分别免疫Balb/c鼠,并通过免疫组化检测表达产物.通过ELISA 和体外疟原虫生长抑制实验来鉴定DNA免疫所诱导的保护性免疫反应.结果扩增到特异的9个Pf332基因片段,并正确插入pMD-18T载体.序列测定和拼接结果显示, 恶性疟原虫FCC1/HN株Pf332基因长16 377bp,编码5 458个氨基酸,相对分子质量约615.28 ku.Ag332包含17个高度简并的富谷氨酸重复序列,抗原中谷氨酸占30.18%.恶性疟原虫FC C1/HN株和3D7株Ag332的氨基酸残基同源性达94.55%.免疫组化检测显示R0、R1和R2在鼠肌肉组织中表达.分别免疫了pcDNA3-S-R0、pcDNA3-S-R1和pcDNA3-S-R2的实验组IgG含量显著大于空白对照组和pcDNA3组(P＜0.05).体外疟原虫生长抑制实验结果表明,pcDNA3-S-R0、pcDNA3-S-R1和pcDNA3-S-R2组的免疫血清在1:5稀释度时对恶性疟原虫的生长有抑制作用.结论 Pf332抗原是一包含很多高度简并的富谷氨酸重复序列的大蛋白,Pf332基因片段R1和R2编码潜在的抗原表位重复序列.
目的瞭解噁性瘧原蟲FCC1/HN株Pf332抗原(Ag332)的初級結構和潛在的抗原錶位.方法根據Pf332基因已知序列,閤成9對引物用于從噁性瘧原蟲FCC1/HN株基因組DNA 中擴增Pf332基因片段.將擴增得到的9箇Pf332基因片段插入pMD-18T載體後測序.用DNAstar軟件將測定的Pf332基因片段序列拼接齣成完整的 Pf332基因序列.分彆用SAPS、Tmpred、SingalP 和Blastn程序來分析Ag332的初級結構和序列同源性.將與Pf332基因的9595～10083、10339～10767 和10855～11247位堿基對應的R0 、R1和 R2片段分彆插入真覈錶達載體pcDNA3-S.將pcDNA3-S-R0、pcDNA3 -S-R1 和pcDNA3-S-R2分彆免疫Balb/c鼠,併通過免疫組化檢測錶達產物.通過ELISA 和體外瘧原蟲生長抑製實驗來鑒定DNA免疫所誘導的保護性免疫反應.結果擴增到特異的9箇Pf332基因片段,併正確插入pMD-18T載體.序列測定和拼接結果顯示, 噁性瘧原蟲FCC1/HN株Pf332基因長16 377bp,編碼5 458箇氨基痠,相對分子質量約615.28 ku.Ag332包含17箇高度簡併的富穀氨痠重複序列,抗原中穀氨痠佔30.18%.噁性瘧原蟲FC C1/HN株和3D7株Ag332的氨基痠殘基同源性達94.55%.免疫組化檢測顯示R0、R1和R2在鼠肌肉組織中錶達.分彆免疫瞭pcDNA3-S-R0、pcDNA3-S-R1和pcDNA3-S-R2的實驗組IgG含量顯著大于空白對照組和pcDNA3組(P＜0.05).體外瘧原蟲生長抑製實驗結果錶明,pcDNA3-S-R0、pcDNA3-S-R1和pcDNA3-S-R2組的免疫血清在1:5稀釋度時對噁性瘧原蟲的生長有抑製作用.結論 Pf332抗原是一包含很多高度簡併的富穀氨痠重複序列的大蛋白,Pf332基因片段R1和R2編碼潛在的抗原錶位重複序列.
목적료해악성학원충FCC1/HN주Pf332항원(Ag332)적초급결구화잠재적항원표위.방법근거Pf332기인이지서렬,합성9대인물용우종악성학원충FCC1/HN주기인조DNA 중확증Pf332기인편단.장확증득도적9개Pf332기인편단삽입pMD-18T재체후측서.용DNAstar연건장측정적Pf332기인편단서렬병접출성완정적 Pf332기인서렬.분별용SAPS、Tmpred、SingalP 화Blastn정서래분석Ag332적초급결구화서렬동원성.장여Pf332기인적9595～10083、10339～10767 화10855～11247위감기대응적R0 、R1화 R2편단분별삽입진핵표체재체pcDNA3-S.장pcDNA3-S-R0、pcDNA3 -S-R1 화pcDNA3-S-R2분별면역Balb/c서,병통과면역조화검측표체산물.통과ELISA 화체외학원충생장억제실험래감정DNA면역소유도적보호성면역반응.결과확증도특이적9개Pf332기인편단,병정학삽입pMD-18T재체.서렬측정화병접결과현시, 악성학원충FCC1/HN주Pf332기인장16 377bp,편마5 458개안기산,상대분자질량약615.28 ku.Ag332포함17개고도간병적부곡안산중복서렬,항원중곡안산점30.18%.악성학원충FC C1/HN주화3D7주Ag332적안기산잔기동원성체94.55%.면역조화검측현시R0、R1화R2재서기육조직중표체.분별면역료pcDNA3-S-R0、pcDNA3-S-R1화pcDNA3-S-R2적실험조IgG함량현저대우공백대조조화pcDNA3조(P＜0.05).체외학원충생장억제실험결과표명,pcDNA3-S-R0、pcDNA3-S-R1화pcDNA3-S-R2조적면역혈청재1:5희석도시대악성학원충적생장유억제작용.결론 Pf332항원시일포함흔다고도간병적부곡안산중복서렬적대단백,Pf332기인편단R1화R2편마잠재적항원표위중복서렬.
Objective To understand the primary structure and potential antigenic epitopes of antigen Pf332(Ag332) of P.falciparum iso late FCC1/HN.Methods Based on the published Pf332 gene sequence , nine pairs of primers were designed for the PCR amplification of the Pf332 gen e fragments from genomic DNA of P.falciparum isolate FCC1/HN. The amplified gene fragments were subcloned into pMD-18T vectors and sequenced. The sequences were aligned using DNAstar software to obtain the full-length sequence of the gene Pf332. The primary structure and sequence homology of Ag332 were analyzed by SAPS, Tmpred, SingalP and Blastn programs. Three fragments, R0, R1 and R2, cor responding to nt#9595-10083, nt#10339-10767 and nt#10855-11247 of Pf332 gene were subcloned into the eukaryotic expression vector pcDNA3-S separately. The Balb/c mice were immunized with pcDNA3-S-R0, pcDNA3-S-R1 and pcDNA3- S-R2 separately, and the expressions of the recombinant proteins were detected by immunohistochemistry assay. The protective immune responses elicited by DNA I mmunization were analyzed by ELISA and parasite growth inhibition tests in vitro .Results Nine Pf332 gene fragments were specifically amplif ied, subcloned into pMD-18T vectors and sequenced. Pf332 gene of the P.falci parum isolate FCC1/HN was 16,377 bp in length, encoding a protein of 5,458 ami no acids, about 615.28kDa. The Ag332 contains 17 regions of highly degenerated Glu-rich repeats, with 30.18% Glu in total amino acids of Ag332. Ag332 of P.falciparum isolate FCC1/HN and 3D7 exhibited 94.55 % homology in amino acid residues. The results of immunohischemistry assay showed that R0, R1 and R2 were expressed in mice muscle tissue. The amount of IgG antibody of the groups immu nized with pcDNA3-S-R0, pcDNA3-S-R1 and pcDNA3-S-R2 were higher than those of blank and pcDNA3 groups (P＜0.05). The result of parasite growth inhibition test showed that the immunized sera at 1∶5 dilution of groups of pcDNA3-S-R0, pcDNA3-S-R1 and pcDNA3-S-R2 had an incomplete inhibitor y effect on P.falciparum growth. Conclusion The antigen Pf332 is an large protein containing highly degenerated Glu-rich repeats. Pf332 gene fragments, R1 and R2 encoding potent antigenic epitope repeats.
