3 . the effect of sporulation - independent promotor on toxicity of natural strain in order to study the effect of sporulation - independent promotor ( p3a ) , p3a was spliced with the cry1c gene , then inserted into the shuttle vector pht304 , and then recombinated plasmid pbmb827 was obtained . after transferring pbmb827 into strain ybt - 1520 , it was surprising that the transformants had almost no potency against all lepidopteran larvae tested 3非依赖芽胞形成icp的cry3a启动子( p _ ( 3a ) )对野生菌株特性的影响带p _ ( 3a )和cry1c基因的重组质粒pbmb827转入ybt - 1520 ，转化子对所测昆虫的毒力下降非常明显，芽胞和晶体也很难脱落。
In the pqe60 - hyuh , the hyuh is singly controlled by the t5 promotor , while in pt221 - hyuh the hyuh is co - expressed with a molecular chaperon . the expression products of recombinant plasmid were further analysis in solubility and activity . a protein band about 50kd was detected by sds - page in the recombinant cell lysate 将pcr扩增得到的hyuhdna片段分别置于载体质粒pqe60的t5启动子和pt221的t7启动子下游，构建了单独表达质粒pqe60 - hyuh和融合表达质粒pt221 - hyuh ，并对两种重组质粒的表达产物进行了可溶性分析和活性测定。
Then , 5 . 5kb thrombiotin gene was amplified with the same technique from the genome of a baby ' s blood , which included the begining part of intronl to the teminator . in addition , 6 . 0kb and 1 . 8kb homlogous arms were also amplified from a cow with high yield . the 6 . 0kb homologous arm contains the promotor , extron 1 , extron2 , extron3 and intron 1 , intron2 and part of the intron3 fragment , while the 1 . 8kb homologous right arms contains exon13 , exon14 and part of intron 13 , the whole intron14 and part intron 14 of asl - casein gene of bovine 通过长片段pcr从高产奶牛的基因组中获得了打靶所需的长、短同源臂序列，长度分别为6 . 0kb和1 . 8kb ，位于s1 -酪蛋白基因的5上游区到第三内含子和十二到十四内含子；从绵羊全血基因组克隆得到了绵羊的-酪蛋白基因启动子区到第二内含子区4 . 1kb的5调控序列；利用同对引物克隆得到了水牛的同基因序列；从广西当地一婴儿脐血基因组中通过获得了人血小板生成素基因，位于第1内含子到终止子后部分的序列，长达5 . 5kb 。
The hwtx - i gene was chemically synthesized according to its known cdna sequence , the gene was inserted into vector ppic9k which contained aoxj promotor and the sequence of a secreting signal peptide - a - factor , the cloning ppic9k / hwtx - i was constructed and confirmed by two - step pcr and dna sequence analysis , then it was transformed into host strain gs115 , a his + muts cell line was screened and multicopy transformants were screened by various g418 concentrations , the multicopy transformant was named gh1 . gh1 was cultivated in flasks . after 6 days of induction by 0 . 5 % methanol , the supernatant was checked by 16 . 5 % tricine - sds page , which showed there was a band in the position of 3 . 5 - 6 . 1kd , then it was isolated and desalted by ultrofiltration followed by ion exchange of cm column , after reverse phase hplc of ci8 and vacuum drying , the purified rhwtx - 1 was obtained which was proved to be correct recombinant hwtx - i by tricine sds - page , maldi - tof mass spectrometry , amino acid composition analysis , the n - terminal amino acid sequence and its biological activity , the final field of the purified rhwtx - i was about 80mg / l , accounting for 23 . 6 % of it total secretory proteins 将带有hwtx -基因的ppic9k经blgii线性化后，转化酵母宿主菌gs115原生质体后经筛选阳性克隆并经表型鉴定为his ~ + mut ~ s酵母菌，进一步用遗传毒素g418筛选多拷贝的转化菌株，命名为gh1 ；将gh1甲醇酵母菌用0 . 5的甲醇诱导表达，发酵上清经90饱和度的( nh _ 4 ) _ 2so _ 4沉淀， yw - 3 ( mwc03000 )的超滤膜超滤，再经cm阳离子交换， c _ ( 18 )反相hplc纯化得到分子量为4kd左右的组分，其中4289 . 05的组分经质谱鉴定，氨基酸组成分析和序列测定为正确的表达产物，生物学活性表明其活性为天然毒素活性70 % ，表达量为80mg / l 。
