中华锯齿米虾卵黄蛋白的纯化及基因克隆
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摘要
中华锯齿米虾(Neocaridina denticulata sinensis)具有生长迅速、生命力顽强、生长周期短、容易饲养等特点,是一种良好的实验材料。目前对中华锯齿米虾卵黄蛋白(vitellin, Vn)的研究比较少,本文对中华锯齿米虾的卵黄蛋白进行了分离纯化,同时采用基因工程方法研究其卵黄蛋白的编码基因,获得其编码框内部分序列,为最终揭示卵黄蛋白的合成机理奠定基础。
卵黄蛋白的分离纯化及部分生化性质：
利用聚丙烯酰胺凝胶电泳,采用不同的染色方法,从成熟中华锯齿米虾卵巢中鉴定出卵黄蛋白,用电泳洗脱的方法对其纯化,并对其部分生化性质进行分析。
研究发现,中华锯齿米虾卵黄蛋白总分子量为438 kDa, SDS-聚丙烯酰胺凝胶电泳分析它由两个亚基组成。同时发现,两个亚基的糖染色反应均成阳性,说明两个亚基上均有糖辅蛋白的结合。样品缓冲液中β-巯基乙醇的有无并不影响亚基分离,推测亚基之间不存在二硫键。
卵黄蛋白的基因工程方法研究：
在解剖中华锯齿米虾卵巢及肝胰腺的基础上,使用TRNzol试剂提取中华锯齿米虾卵黄蛋白总RNA, RT-PCR扩增编码卵黄蛋白基因片段,将所得PCR产物测序,所测得462 bp序列与三种已知甲壳动物卵黄蛋白序列相似性较高,且相似区域都在三种卵黄蛋白的编码框架之内,推测所得序列为中华锯齿米虾卵黄蛋白基因片段。
Neocaridina denticulata sinensis had many advantages, such as, grew fast, vitality stubborn, vegetative cycle deficient and easily to rise, which was a good subject. But the research on the vitellin of N. denticulata sinensis was little, In this dissertation, isolation, purification, and biochemical characterization of the vitellin from the mature ovary of N. denticulata sinensis. In this paper, genetic engineering technique was used for studying on the vitellin of N. denticulata sinensis, obtaining a part of open reading frames of it and established the theoretical basis on the synthesis mechanism of vitellin.
The purification and some biochemical characteristic of vitellin:
With the PAGE, still applying various staning methods, our study proved vitellin in the mature ovary of N. denticulata sinensis. According to this paper, the vitellin was purified by electric-elution. And an nanlysis was carried to its partial biochemical characterization.
During the research, the molecular weight of the vitellin was 438 G in a gradient native PAGE. Two subunits in purified vittllin were detected with SDS-PAGE. In the mean time, we found these two subunits all contained carbohydrate moieties based on the staining with periodic acid-Schiff's reagent. Two subunits of vitellin were also observed in SDS-PAGE whether the vitellin was reduced with P-mercaptoethanol or not. So, we supposed that the disulfide bonds was no-existence among the polypeptie subunits.
Study on vitellin with the genetic engineering technique:
During the experiment, ovary and hepatopancreas of N. denticulata sinensis were dissected firstly. Then total RNA was extracted by using the TRIzol reagent. A part of vitellin cDNA fragments in open reading frames was obtained after RT-PCR. After DNA sequencing, 438 bp sequence has relatively high similarity with three kinds of vitellin which has been known. The similar region is in the vitellin open reading frames. The DNA fragments was presumed to a part of N. denticulata sinensis' vitellin gene.
卵黄蛋白的分离纯化及部分生化性质：
利用聚丙烯酰胺凝胶电泳,采用不同的染色方法,从成熟中华锯齿米虾卵巢中鉴定出卵黄蛋白,用电泳洗脱的方法对其纯化,并对其部分生化性质进行分析。
研究发现,中华锯齿米虾卵黄蛋白总分子量为438 kDa, SDS-聚丙烯酰胺凝胶电泳分析它由两个亚基组成。同时发现,两个亚基的糖染色反应均成阳性,说明两个亚基上均有糖辅蛋白的结合。样品缓冲液中β-巯基乙醇的有无并不影响亚基分离,推测亚基之间不存在二硫键。
卵黄蛋白的基因工程方法研究：
在解剖中华锯齿米虾卵巢及肝胰腺的基础上,使用TRNzol试剂提取中华锯齿米虾卵黄蛋白总RNA, RT-PCR扩增编码卵黄蛋白基因片段,将所得PCR产物测序,所测得462 bp序列与三种已知甲壳动物卵黄蛋白序列相似性较高,且相似区域都在三种卵黄蛋白的编码框架之内,推测所得序列为中华锯齿米虾卵黄蛋白基因片段。
Neocaridina denticulata sinensis had many advantages, such as, grew fast, vitality stubborn, vegetative cycle deficient and easily to rise, which was a good subject. But the research on the vitellin of N. denticulata sinensis was little, In this dissertation, isolation, purification, and biochemical characterization of the vitellin from the mature ovary of N. denticulata sinensis. In this paper, genetic engineering technique was used for studying on the vitellin of N. denticulata sinensis, obtaining a part of open reading frames of it and established the theoretical basis on the synthesis mechanism of vitellin.
The purification and some biochemical characteristic of vitellin:
With the PAGE, still applying various staning methods, our study proved vitellin in the mature ovary of N. denticulata sinensis. According to this paper, the vitellin was purified by electric-elution. And an nanlysis was carried to its partial biochemical characterization.
During the research, the molecular weight of the vitellin was 438 G in a gradient native PAGE. Two subunits in purified vittllin were detected with SDS-PAGE. In the mean time, we found these two subunits all contained carbohydrate moieties based on the staining with periodic acid-Schiff's reagent. Two subunits of vitellin were also observed in SDS-PAGE whether the vitellin was reduced with P-mercaptoethanol or not. So, we supposed that the disulfide bonds was no-existence among the polypeptie subunits.
Study on vitellin with the genetic engineering technique:
During the experiment, ovary and hepatopancreas of N. denticulata sinensis were dissected firstly. Then total RNA was extracted by using the TRIzol reagent. A part of vitellin cDNA fragments in open reading frames was obtained after RT-PCR. After DNA sequencing, 438 bp sequence has relatively high similarity with three kinds of vitellin which has been known. The similar region is in the vitellin open reading frames. The DNA fragments was presumed to a part of N. denticulata sinensis' vitellin gene.
引文
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