The tk gene contained an open reading frame of 957 bp encoding a 318 aa protein . upstream of the tk orf , three putative gc boxes are located at positions - 22 , - 166 and - 199 . a potential poly a signal begins 110 nucleotides downstream from the termination codon at position 1306 在tkorf上游- 22 、 - 166 、 - 199位存在3個gc框樣序列,在終止密碼子下游第110個核苷酸處的1306位存在有多聚腺苷加尾信號。
Three pairs of primers were designed according to the sequences pulished by the genebank in order to amplifiy gd , ge and tk gene of the pseudorabies virus min - a strain . the gd , ge and tk gene were obtained by polymerase chain reaction ( pcr ) , and then cloned into the pgem - t easy vector 參考genebank收錄的偽狂犬病病毒gd 、 ge 、 tk基因的序列設計了三對引物,對prvmin - a株進行了pcr擴增,擴增產物克隆于pgem - teasy載體。
In this study , a 1 . 7kb kpni fragment and a lacz gene expression cassette carrying the e . coli lacz gene under the control of sv40 promoter were inserted into the transfer vector pbdtk - uni ( a 277bp acci - accl fragment in the tk gene was deleted ) . the new transfer vector was called puni - lacz . the transfer vector puni - lacz and purified genomic dna of strain bartha - k61 were used to cotransfect vero cells using lipofectin transfection procedure 本研究以呈ge ~ -表型的經典弱毒疫苗bartha - k61株為親本株,在通用prv轉移載體pbdtk - uni的基礎上,在其多克隆位點中插入由sv40早期啟動子控制下的lacz基因表達盒,同時將下游同源臂增加了一個1 . 7kb的kpni片段,使上下游同源臂的長度都超過了1kb ,構建了一個新的轉移載體puni - lacz 。
Was multiplied and the tk gene was cloned . the cloned tk gene was retrieved by proper restrictive hemodynamics . the retrieved tk gene was labeled by digoxin according to the kit of labeling and detection of digoxin . then , the specificity and sensitivity of tk gene probe were detected with dot blot hybridization . the sequence of tk gene of nm98a strain was analysed . the result of the analysis of tk gene ' s sequence confirmed that autoploidy between tk gene of nm - 98a strain and issued strain was 99 . 7 % 本研究中首次對iltv - nm98a株的tk基因進行了克隆和序列分析,結果表明: iltv - nm98a株tk基因的核苷酸序列與已發表的iltvtk基因的核苷酸序列具有高度的同源性,兩者之間僅相差4個核苷酸,同源性高達99 . 7 ,從而證實了iltvtk基因是高度保守的,為iltvtk基因核酸探針的制備提供了有力的依據。
In this study , iltv - nm98a strain and iltv - wanggang strain were multiplied in chorioallantois . a pair of primers were devised according to the nucleic acid sequence of iltv tk gene and the dna of multiplied virus was used as pattern to amplify the gene of tk by polymerase chain reaction ( pcr ) . the product of pcr was linked with suitable plasmid . then , the recombined plasmid was converted to escherichia coli . the converted escherichia coli 根據已發表的iltvtk基因的核苷酸序列設計一對pcr引物,以增殖的兩株iltv的dna為模板,分別對它們的tk基因進行pcr擴增。將回收的pcr產物連接到適當的質粒載體上,轉化感受態大腸桿菌,通過篩選對iltvtk基因的陽性克隆進行擴增培養。