CAJ | 학술논문

以旋毛虫新生幼虫期(NBL)脱氧核糖核酸酶-Ⅱ(DNaseⅡ)基因家族中全长序列T31D4为研究对象,对其融合蛋白的核酸酶活性进行鉴定并摸索最佳酶切条件;通过序列分析预测其关键活性氨基酸位点,同时利用基因点突变技术对所预测的位点进行验证.结果表明:旋毛虫的DNaseⅡ具有核酸酶酶切活性,最佳酶切pH为5.0,同时发现其在中性(pH 7.0)及偏碱性(pH 8.0)下仍具有活性.利用Maga2软件对包括旋毛虫在内的以及业已发现的来自38个物种的DNaseⅡ的94个氨基酸序列进行比对,发现一个所有物种DNaseⅡ均高度保守的组氨酸位点,该结果与目前国际上所预测的DNaseⅡ关键活性氨基酸位点不同,目前国际上所预测的组氨酸位点在旋毛虫的DNaseⅡ序列中并非组氨酸(H),而是丝氨酸(S)或赖氨酸(K);通过点突变技术进一步验证这个组氨酸位点才是所有物种DNaseⅡ真正的活性氨基酸位点.同时推测旋毛虫的DNaseⅡ可能为一种新类型的DNaseⅡ.
이선모충신생유충기(NBL)탈양핵당핵산매-Ⅱ(DNaseⅡ)기인가족중전장서렬T31D4위연구대상,대기융합단백적핵산매활성진행감정병모색최가매절조건;통과서렬분석예측기관건활성안기산위점,동시이용기인점돌변기술대소예측적위점진행험증.결과표명:선모충적DNaseⅡ구유핵산매매절활성,최가매절pH위5.0,동시발현기재중성(pH 7.0)급편감성(pH 8.0)하잉구유활성.이용Maga2연건대포괄선모충재내적이급업이발현적래자38개물충적DNaseⅡ적94개안기산서렬진행비대,발현일개소유물충DNaseⅡ균고도보수적조안산위점,해결과여목전국제상소예측적DNaseⅡ관건활성안기산위점불동,목전국제상소예측적조안산위점재선모충적DNaseⅡ서렬중병비조안산(H),이시사안산(S)혹뢰안산(K);통과점돌변기술진일보험증저개조안산위점재시소유물충DNaseⅡ진정적활성안기산위점.동시추측선모충적DNaseⅡ가능위일충신류형적DNaseⅡ.
The characterization of DNase Ⅱ from Trichinella spiralis (T.spiralis) was identified by expression of a full length DNase Ⅱ cDNA termed T31D4-N5 from NBL DNase Ⅱ family,the nucleic acid enzymatic activity and optimal enzyme activity conditions of its fusion protein were identified.The critical activity site of active amino acids of the enzyme was predicted and verified by sequence analysis and point mutations technology.The result indicated the optimal pH of it was pH 5.0 and the enzyme activity still remained at pH 7.0 even 8.0.The critical activity site of active amino acids of the enzyme was predicted by the sequence alignments with software Maga 2,total 94 available DNase Ⅱ sequences from 38 bio-species,a high conserved histidine was found in N-terminal of all available DNase Ⅱs,which was different from previous reported sites which were not histidine(H),but serine (S) or lysine (K) in T.spiralis DNase Ⅱ amino acid sequence,this predicted site was also confirmed by point mutation.Concerning the unique characteristics of T.spiralis DNase Ⅱ,it is most probably a new type DNase Ⅱ.