利用cDNA末端快速扩增技术克隆出凡纳滨对虾(Litopenaeus vannamei)核自身抗原精子蛋白(NASP)基因,NASP基因c DNA全长2258 bp,其中包括92 bp 5′端非编码区,147 bp 3′端非编码区及2019 bp开放阅读区,编码673个氨基酸,预测分子量为74.18...利用cDNA末端快速扩增技术克隆出凡纳滨对虾(Litopenaeus vannamei)核自身抗原精子蛋白(NASP)基因,NASP基因c DNA全长2258 bp,其中包括92 bp 5′端非编码区,147 bp 3′端非编码区及2019 bp开放阅读区,编码673个氨基酸,预测分子量为74.18 k D。该序列已提交至Gen Bank,序列号为KT274811。在NCBI上进行序列比对后发现,其与斑节对虾(Penaeus monodon)NASP序列相似性最高,为90%。采用荧光定量PCR方法测定了不同发育时期卵巢与肝胰腺中NASP m RNA相对表达水平,结果表明,NASP在卵巢中的表达量高于肝胰腺中的表达量,且在Ⅱ期表达量最高,Ⅲ期表达量最低。此外,通过构建系统进化树比较了凡纳滨对虾NASP与其他物种间的遗传距离。实验结果为进一步研究该基因在凡纳滨对虾卵巢发育中的作用提供了依据。展开更多
To study whether the antibody against the testis form of the nuclear autoantigenic sperm protein(tNASP)could result in reproductive failure,we successfully cloned and expressed a 339-bp cDNA fragment of mouse tNASP(mt...To study whether the antibody against the testis form of the nuclear autoantigenic sperm protein(tNASP)could result in reproductive failure,we successfully cloned and expressed a 339-bp cDNA fragment of mouse tNASP(mtNASP).Using mouse as a model,recombinant mtNASP(rmtNASP)and a synthetic peptide,human tNASP393-408(htNASP393-408),were investigated for their antifertility effect.Active immunization with rmtNASP or the synthesized peptide raised high antibody titers in the immunized mice.Sperm-egg binding and fusion assay were carried out in 8-10-week-old BALB/c mice.Sperm-egg binding and in vitro fertilization of mouse oocytes were inhibited by co-incubation of zona-free mouse oocytes with capacitated mouse spermatozoa in the presence of varying concentrations of the antisera against rmtNASP.There was a significant antifertility effect in animals immunized with rmtNASP or the synthesized peptide.The effect on fertility in the mice immunized with the synthesized peptide was reversible.Our data indicate that active immunization with rmtNASP antigen may induce a strong antibody response that causes an inhibition of fertility.展开更多
文摘利用cDNA末端快速扩增技术克隆出凡纳滨对虾(Litopenaeus vannamei)核自身抗原精子蛋白(NASP)基因,NASP基因c DNA全长2258 bp,其中包括92 bp 5′端非编码区,147 bp 3′端非编码区及2019 bp开放阅读区,编码673个氨基酸,预测分子量为74.18 k D。该序列已提交至Gen Bank,序列号为KT274811。在NCBI上进行序列比对后发现,其与斑节对虾(Penaeus monodon)NASP序列相似性最高,为90%。采用荧光定量PCR方法测定了不同发育时期卵巢与肝胰腺中NASP m RNA相对表达水平,结果表明,NASP在卵巢中的表达量高于肝胰腺中的表达量,且在Ⅱ期表达量最高,Ⅲ期表达量最低。此外,通过构建系统进化树比较了凡纳滨对虾NASP与其他物种间的遗传距离。实验结果为进一步研究该基因在凡纳滨对虾卵巢发育中的作用提供了依据。
基金the National Natural Science Foundation of China(No.30470909 to Chen Xu,and No.30640031 to Chen Xu)the Deng Shan Project of Shanghai Municipal Commission of Science and Technology(No.06JC14046 to Chen Xu)+2 种基金the‘973’Basic Research Funding Scheme of China(No.G199905501 to Chen Xu)the Science Research Project of Science&Technology Commission of Shanghai(No.06ZR14057 to Jian-Li Shi)the Shanghai Leading Academic Discipline Project(No.S30201 to Chen Xu).
文摘To study whether the antibody against the testis form of the nuclear autoantigenic sperm protein(tNASP)could result in reproductive failure,we successfully cloned and expressed a 339-bp cDNA fragment of mouse tNASP(mtNASP).Using mouse as a model,recombinant mtNASP(rmtNASP)and a synthetic peptide,human tNASP393-408(htNASP393-408),were investigated for their antifertility effect.Active immunization with rmtNASP or the synthesized peptide raised high antibody titers in the immunized mice.Sperm-egg binding and fusion assay were carried out in 8-10-week-old BALB/c mice.Sperm-egg binding and in vitro fertilization of mouse oocytes were inhibited by co-incubation of zona-free mouse oocytes with capacitated mouse spermatozoa in the presence of varying concentrations of the antisera against rmtNASP.There was a significant antifertility effect in animals immunized with rmtNASP or the synthesized peptide.The effect on fertility in the mice immunized with the synthesized peptide was reversible.Our data indicate that active immunization with rmtNASP antigen may induce a strong antibody response that causes an inhibition of fertility.
