人载脂蛋白APOA5的克隆表达纯化及相互作用蛋白研究

人类的载脂蛋白A5（apolipoprotein A5，APOA5）是一个新近发现的载脂蛋白家族成员。它在血浆中的含量比其他载脂蛋白低1-2个数量级，但能显著影响血浆三酰甘油水平，对血脂代谢具有重要意义，可以作为降血脂药物治疗中一个强有力的潜在靶标。 由于APOA5在血浆中含量低，直接从血浆中分离纯化很困难，国内一直没有报道简易可靠的纯化方法。为进一步研究APOA5的生物学特性，探讨其与TG代谢中的其它关键成分之间的相互关系，揭示其在脂类代谢相关疾病中的重要地位，必须有大量的蛋白和抗体用于基础研究。因此本研究首先利用基因工程技术，诱导表达纯化APOA5蛋白，免疫动物制备多克隆抗体，为进一步研究人肝脏细胞中APOA5的相互作用蛋白，研究APOA5蛋白在肝脏细胞中的功能奠定基础。 为了深入研究APOA5在肝脏中如何行使功能，我们采用细菌双杂交技术寻找与APOA5相互作用的蛋白因子。并采用Pull-down技术，免疫荧光及免疫共沉淀技术进一步确证其在体外和体内的相互作用关系，为进一步阐明APOA5在体内的生理功能提供了新的线索。 第一部分 APOA5基因的克隆、原核表达、纯化及其多克隆抗体的制备 本研究首先应用基因克隆技术，从人肝癌细胞系SMMC-7721的cDNA中扩增出1.1 kb的ApoA5基因全长序列。然后将其克隆至表达载体pThioHisD，构建原核表达载体pTH-APOA5。该重组质粒转化至大肠杆菌 BL21（DE3），成功实现人APOA5融合蛋白在大肠杆菌中的表达。经发酵得到高效表达的融合蛋白。 融合蛋白在 IPGT 诱导下以包涵体的形式大量表达。利用融合蛋白上的一段组氨酸序列，用镍离子亲和柱进行纯化和复性后，获得较高纯度的人APOA5融合蛋白。利用该融合蛋白免疫新西兰大耳白兔，获得了高效价的兔抗人APOA5多克隆抗体，Western Blot结果显示此多克隆抗体与APOA5特异性结合。 第二部分 细菌双杂交筛选与APOA5相互作用的蛋白 本实验首先构建了pBT-APOA5重组质粒，经双酶切、PCR和测序鉴定证明重组诱饵质粒构建成功，并进行了表达、自激活鉴定。Western Blot鉴定证实报告菌株中表达了分子量为 68 kD左右的重组融合蛋白，与预测的分子量APOA5（41 kD）/lamda cI （27 kD）一致。自激活实验证明诱饵蛋白不能单独激活报告基因，可用于筛选人肝脏cDNA文库。经过双重抗性筛选和回复筛选，分离出10个阳性克隆。对结果进行生物信息学分析，得到7个与APOA5相互作用的蛋白，其中BI1为细胞凋亡调节因子；ATP6、CYTB、ND2、COX-1为线粒体表达蛋白； ALB、TTR为血清蛋白。 第三部分 APOA5与BI1相互作用的确证 首先构建了BI1的原核表达载体pGEX-5X-3-BI1，利用Pull-down实验检测了APOA5与BI1在体外具有相互作用。然后构建了BI1的真核表达载体pCDNA3.1-HA-BI1和APOA5的真核表达载体pCDNA3.1-APOA5，并验证其表达。通过免疫荧光细胞内共定位研究发现，靶蛋白APOA5主要分布于胞浆，与BI1在HEK293细胞有共定位，即APOA5与BI1存在相互作用的可能。最后利用免疫共沉淀手段，在HEK293细胞中确证了靶蛋白APOA5与BI1在体内的相互作用。 上述研究结果，为深入研究APOA5在体内的生物学功能提供了新的思路。 Apolipoprotein A5 (APOA5) is a newly discovered protein belongs to apolipoprotein family. APOA5’s concentration is 1-2 orders of magnitude lower than other apolipoproteins in the circulation. APOA5 significantly affected plasma triglyceride levels, which is important on lipid metabolism. APOA5 has strong potential to be used as a hypolipidemic drug target. Large amount of APOA5 protein and antibodies are needed in basic research, such as biological characteristics study of the APOA5, its relationship with other key components in TG metabolism, its role played in Lipid metabolism-related diseases. Due to its low concentration in plasma, separation and purification of APOA5 from the plasma is very difficult. Until now no report on simple and reliable method for purification has been published in China. In this study, we firstly got APOA5 recombinant protein using genetic engineering technology. The purified recombinant protein was used to immunize rabbits to get antiserum. It is important for further study of the APOA5 protein-interacting protein. And it lays the foundation for studing APOA5 function in liver. In order to study APOA5 function in liver, we used bacterial two-hybrid technology to find the APOA5 protein interactor. Pull-down, immunofluorescence and immunoprecipitation techniques were used to further confirm the interaction between APOA5 with its interactor in vitro and in vivo. All of these stdudies provided new clues on its physiological functions in vivo. Part I: Cloning, prokaryotic expression, purification and polyclonal antibody preparation of APOA5 First of all, we amplified APOA5 CDS sequence from the human hepatoma cell line SMMC-7721, and subcloned into Expression vector pThioHisD, and got the recombinants named pTH-APOA5. The plasmid was transformed to BL21 (DE3). E. coli BL21(DE3) cells bearing the pTH-APOA5 plasmid were cultured and APOA5 protein synthesis was induced by the addition of IPTG. Recombinant protein was expression in the form of inclusion. Inclusion bodies were dissolved in phosphate-buffered saline containing 8 M urea and 40 mM imidazole, then applied to a Ni2+ affinity column, and were eluted in a buffer containing 4 M urea and 200 mM imidazole. Fractions containing the APOA5 protein were pooled and dialyzed against buffer containing phosphate-buffered saline. Antiserum to recombinant human APOA5 was generated by immuning rabbit. Western Blot showed that this antiserum specific binding with APOA5. Part II Two-hybrid system screening protein interactions with the APOA5 The coding sequence of human APOA5 was amplified using synthetic oligonucleotide primers from pTH-APOA5 vector and was subcloned into the pBT plasmidc to yield pBT-APOA5 vector. DNA sequencing was performed to verify that no unwanted mutations occurred during the process of plasmid vector construction. We verified recombinant protein expression and tested self-activation by pBT-APOA5 prior to screening. Western Blot verified inducing a 68 kD band, consistent with the predicted molecular weight (APOA5 41 kD, lamda cI 27 kD). pBT-APOA5 can be used for screening human liver cDNA library because it can not self-activation. Totally 10 positive clones were isolated. The nucleotide sequence of the positive clones were determined and compared to NCBI nucleotide sequence databases. We got 7 protein which interact with APOA5, included BI1(Apoptosis regulator); ATP6, CYTB, ND2, COX-1(Mitochondrial protein) and ALB, TTR(Serum protein). Part III Confirming of interaction between APOA5 with BI1 pGEX-5X-3-BI1 vector was subcloned at first. Pull-down experiments were used to detect the interaction between APOA5 with BI1 in vitro. Later, pCDNA3.1-HA-BI1 and pCDNA3.1-APOA5 were subcloned. Through immunofluorescence co-localization study, we found APOA5 mainly distributed in the cytoplasm. APOA5 is co-localization with BI1 in HEK293 cells. Finally, we verified interaction between APOA5 with BI1 in vivo through immunoprecipitation.

Towards manipulation of post-biosynthetic events in secondary metabolism of plant cell cultures

Plant cell cultures have been suggested as a feasible technology for the production of a myriad of plant-derived metabolites. However, commercial application of plant cell culture has met limited success with only a handful of metabolites produced at the pilot- and commercial-scales. To improve the production of secondary metabolites in plant cell cultures, efforts have been devoted predominantly to the optimization of biosynthetic pathways by both process and genetic engineering approaches. Given that secondary metabolism includes-the synthesis. metabolism and catabolism of endogenous compounds by the specialized proteins, this review intends to draw attention to the manipulation and optimization of post-biosynthetic events that follow the formation of core metabolite structures in biosynthetic pathways. These post-biosynthetic events-the chemical and enzymatic modifications, transport, storage/secretion and catabolism/degradation have been largely unexplored in the past. Potential areas are identified where further research is needed to answer fundamental questions that have implications for advanced bioprocess design. Anthocyanin production by plant cell cultures is used as a case study for this discussion, as it presents a good example of compounds for which there are extensive research publications but still no commercial bioprocess. It is perceived that research on post-biosynthetic processes may lead to future opportunities for significant advances in commercial plant cell cultures. (C) 2002 Elsevier Science Inc. All rights reserved.

Transforming kelp into a marine bioreactor

The past decade has seen the genetic engineering of various types of seaweed. To date, genetic transformation studies have been carried out in several seaweeds, including the red seaweeds Porphyra, Gracilaria, Grateloupia, Kappaphyclus and Ceramium and the green seaweed Ulva. A genetic transformation model system has been established in the most commonly cultivated seaweed, the brown seaweed Laminaria japonica (kelp), based on the transfer of technology used in land plant transformation and also by modulating the seaweed life cycle. This model showed the potential for application of transgenic kelp to the production of valuable products and an indoor cultivation system for transgenic kelp was proposed, taking into account necessary factors for bio-safety. In this review, the establishment at use of the kelp transformation model is introduced, highlighting the potential for transforming kelp into a marine bioreactor.

Molecular biotechnology of marine algae in China

Molecular biotechnology of marine algae is referred to as the biotechnology on the identification, modification, production and utilization of marine algal molecules. It involves not only the manipulation of macromolecules such as DNA, RNA and proteins, but also deals with low molecular weight compounds such as secondary metabolites. In the last decade, molecular systematic researches to investigate the relationship and to examine the evolutionary divergence among Chinese marine algae have been carried out by Chinese scientists. For example, RAPD has been widely used in several laboratories to elucidate genetic variations of the reds, such as Porphyra, Gracilaria, Grateloupia and the greens such as Ulva and Enteromorpha. Some important data have been obtained. The study on molecular genetic markers for strain improvement is now in progress. In 1990s, genetic engineering of economic seaweeds such as Laminaria, Undaria, Porphyra, Gracilaria and Grateloupia has been studied in China. For Laminaria japonica, the successfully cultivated kelp in China, a model transformation system has been set up based on the application of plant genetic techniques and knowledge of the algal life history. Progress has been made recently in incorporating a vaccine gene into kelp genome. Evidence has been provided showing the expression of gene products as detectable vaccines. In the present paper, the progress of molecular biotechnological studies of marine algae in China, especially researches on elucidating and manipulating nucleic acids of marine algae, are reviewed.

Expression of hepatitis B surface antigen gene (HBsAg) in Laminaria japonica (Laminariales, Phaeophyta)

A transformation model for Laminaria japonica was established from 1993 to 1998, on the basis of which the transgenic kelp with heterologous gene encoding hepatitis B surface antigen (HBsAg) was obtained by using the micro-particle bombardment transformation method. Results of quantitative ELISA showed that HBsAg in transgenic kelp was 0.529 mug/mg soluble proteins on average and the highest value was 2.497 mug/mg, implying that recombinant HBsAg had natural epitope. Further support for the integration of HBsAg gene into kelp genome was obtained by PCR-Southern and total DNA hybridization. Prospect of kelp bio-reactor producing high value materials such as edible HBV vaccine was discussed as well.

Transient expression of lacZ in bombarded unicellular green alga Haematococcus pluvialis

This paper reports for the first time the transient expression of a reporter gene, LacZ, in the unicellular green alga Haematococcus pluvialis. By employing the micro-particle bombardment method, motile cells in the exponential phase showed transient expression of lacZ. This was detected in bombarded motile cells under the rupture-disc pressures of 3103 KPa and 4137 KPa. Transient expression of LacZ gene could not be observed in non-motile cells of this alga under the same transformation condition. No LacZ background was found in either the motile cells or the non-motile cells. The study suggests a promising potential of the SV40 promoter and the lacZ reporter gene in genetic engineering of unicellular green algae.

Foreign gene transfer into Chinese shrimps (Penaeus chinensis) with gene gun

Plasmids pG DNA-RZ1 with a GFP (green fluorescent protein) reporter gene and a ribozyme gene incising penaeid white spot baculovirus (WSBV) were first introduced into the fertilized eggs of Chinese shrimps by gene gun. The treated and control samples of different development stages were observed with a fluorescent microscope. The transient expression of GFP gene was high in nauplius and zoea larvae. Results from RT-PCR and PCR for adults showed that the foreign genes had been transferred into the shrimps and had expressed the corresponding proteins. This work has established a transgenic method for penaeid shrimps, which will set base for the application of genetic engineering breeding into industry.

Recombinant expression of rt-PA gene (encoding Reteplase) in gametophytes of the seaweed Laminaria japonica (Laminariales, Phaeophyta)

The life cycle of seaweed Laminaria japonica involves a generation alternation between diploid sporophyte and haploid gametophte. The expression of foreign genes in sporophte has been proved. In this research, the recombinant expression in gametophyte was investigated by particle bombardment with the rt-PA gene encoding the recombinant human tissue-type plasminogen activator (Reteplase), which is a thrombolytic agent for acute myocardial infarction (AMI). Transgenic gametophytes were selected by their resistance to herbicide phosphiothricin (PPT), and proliferated in an established bubble column photo-bioreactor. According to the results from quantitative ELISA, Southern blotting, and fibrin agarose plate assay (FAPA) for bioactivity, it was showed that the rt-PA gene had been integrated into the genome of gametophytes of L. japonica, and the expression product showed the expected bioactivity, implying the proper post-transcript modification in haploid gametophyte.

Purification of human tissue prokallikrein excreted from insect cells by liquid chromatography

Tissue kallikrein, generally existing in living bodies as prokallikrein, is a serine proteinase that has proven of great significance to treat hypertension, cardiopathy and nephropathy. Although the extraction of tissue kallikrein from human urine is the most commonly used method to obtain such a protein, not only the yield is very little, but also the procedure is rather complex. Furthermore, the biological safety is uncertain. Therefore, the preparation of such a protein by genetic engineering method, including gene expression, cell culture, separation and purification, is very important. In this paper, a new method to obtain purified tissue prokallikrein excreted from insect cells by liquid chromatography has been proposed. In contrast to the previously published papers, the purification procedure is simplified to only three steps with the final yield of 57% and the purity of 95%, which is not only convenient, but also low-cost and suitable for the large-scale preparation of such a protein. The purified protein is further validated as prokallikrein by high performance liquid chromatography-mass spectrometry and amino acid sequencing. (c) 2005 Elsevier B.V. All rights reserved.