暑期实习报告

2008年暑期实习报告及感想
尹意铭
北京大学生命科学学院
实习所在地：Medical College of Georgia
Augusta, Georgia
USA
实习时间：2008.7—2008.9
Supervisor: Prof. Lin Mei
ErbB protein is a family of single-transmembrane receptor tyrosine kinases which have homology with the EGF receptor. Their ligand, Neuregulin(NRG), contains an epidermal growth factor(EGF)-like domain that signals by stimulating ErbB. Among the ErbB receptors, ErbB4 is the best-characterized for its function in the CNS. Importantly, ErbB4 is the only autonomous NRG1-specific ErbB that can both interact with the ligand and become activated by it as a tyrosine kinase. Neuregulin1 (Nrg1) and its receptor ErbB4 are associated with schizophrenia, a highly debilitating mental disorder.
Several years before I arrived, a yeast two hybrid assay had been conducted by two former lab members to find the potential binding partners of ErbB4, but the positive clones had not been characterized yet. My first job is to identify the positive clones. I revived the positive yeast clones, and got the yeast plasmids. Then I transformed the plasmids to E. coli KC8 strain and make the bacterial plasmids for sequencing. The identification result of totally 37 positive clones is in table 1.
In the 37 clones, 19 of them were unable to be sequenced. Considering that plasmids from KC8 strain may not be suitable for sequencing, I transformed the plasmids to XL1 Blue strain and made the plasmids for sequencing. Still, none of them can be sequenced. Probably the old age of the positive clones influeced the sequencing.
The emergence of so many 14-3-3 proteins attracted my attention. 14-3-3 is highly conserved from bacteria to human and ubiquitously expressed in all human tissues. 14-3-3 is able to bind to many proteins and has a lot of important functions, for example, cell cycle control, apoptosis, stress response. Importantly, 14-3-3 is essential for normal brain development and neuronal migration in the mouse, and some 14-3-3 isoforms are reported to be related to schizophrenia. Therefore，I decided to test the interaction between ErbB4 and 14-3-3 proteins.
Firstly, I tried to see if GST-14-3-3 fusion protein can pull down ErbB4. The pGEX-14-3-3 plasmid is transformed into E. coli BL21 strain. The expression is induced using IPTG (0.7mM) for 3 hours at 37C. The expression of the fusion protein cannot be detected by dyeing with Coomassie brilliant blue, while the sole GST tag expressed well. (Figure 1)
Figure 1: The expression of GST-14-3-3ξ fusion protein. The GST-14-3-3ξ fusion protein cannot be detected by dyeing with Coomassie brilliant blue.
In order to enhance the expression of the fusion protein, I changed the inducing condition of IPTG., using IPTG to induce for 18hours at 25。

C. Still, GST-14-3-3 fusion protein cannot be detected by dyeing. Therefore, I did a Western to check the expression using anti-GST antibody. As the result indicates, all the bacteria transformed with GST-14-3-3 could only express protein with the same molecular weight as GST.( Figure 2 ) As sequencing result shows that the GST-14-3-3 construct is correct, I assume that the 14-3-3 protein is degraded in E.coli.
Figure 2. Western blot of the GST-14-3-3ξ fusion protein.
My next step was to test if 14-3-3 and ErbB4 could co-precipitate. I expressed myc-14-3-3 fusion protein (both Isoform ξand Isoform ζ) and EGFP-ErbB4 fusion protein in human embryonic kidney 293 ( HEK293 ) cell line. The plasmids are transfected using lipofectomine 2000TM. Both of the fusion proteins expressed well ( Figure 3), and a co-IP experiment is conducted. However, the result indicates that myc-14-3-3ξ/ζ cannot co-precipitate with GFP-ErbB4 ( Figure 4).
Figure 3: The expression of pGFP-ErbB4 protein in Hek293 cell.
Considering that the GFP tag is in the intracellular C-terminal of ErbB4 and therefore
highly possible to inhibit the interaction between ErbB4 and other proteins, I used pFlag-ErbB4, in which the Flag tag is in the N terminal of the fusion protein, to test the interaction. I used the kit of jetPEI to transfect Hek293 cells this time. The expression of the protein was good but the co-IP assay still indicated no interaction between 14-3-3 and ErbB4.( Figure 5)
Figure 5: Co-precipitation assay indicates no interaction between myc-14-3-3ξ and two ErbB4 isoforms, CYT1 and CYT2.
In the last two weeks of the internship, I did the subcloning of 15 Wnt genes. These clones will be used to for other assays of one of my collegues, including the verifying of the clustering of these proteins..
实习收获、感想与建议
总的来说，通过这次实习我的收获是巨大的，我认为这个活动应该坚定的进行下去。

在此特别感谢饶院和Dr. Mei, 以及所有为实习活动作出努力的人，你们的高瞻远瞩令人钦佩。

我觉得这次暑期实习活动给我最大的收获在于使我对科研有了更深入系统的了解。

在实践之前，我认为本科生在实验室学习的仅仅是一些技术，认为构建一个质粒或者学会养某种细胞，就是科研。

经过实习，我才感受到真正的科研绝不仅仅是掌握实验技术，而是包括大量的阅读文献、广泛的与同行交流、并通过敏锐的感知力和大胆的想象，提出模型，设计周密的实验和对照等一系列过程。

而实验技术仅仅是期中的一小部分。

这次实习将使我远离过于迷恋实验技术的误区。

这次实习使我认识到了文献阅读的重要性。

在实习的过程中，不论是查找研究对象的相关资料，设计可行的实验方法，还是构建模型，都需要查阅大量的文献。

在实习之前，由于过于关注实验技术，我常忽略了文献阅读的重要性，觉得文献阅读枯燥乏味。

但在实习过程中，我发现文献阅读是十分有用并且有趣的事情，读文献总能让我有茅塞顿开，恍然大悟的感觉，并给我很多启发。

通过在一次Journal Club 上介绍文献，我的公共表达能力也得到了锻炼。

这次暑期实习对我的综合能力有了很大的锻炼。

包括与人交流的能力，英语能力等等。

这次实习使我对文献阅读课产生了一些想法。

我认为文献阅读课是一门非常有意义的课，通过本次实习，我对这一点有了更深入的认识。

同学的阅读动机对于这门课的效果影响很大。

如果同学热爱阅读文献，能积极的阅读思考，那么这门课对同学将有很大的帮助。

反之，如果同学认为文献阅读不重要，或者不是当下最重要的事，那就只是为了这门课而阅读，为了写报告而思考。

因此，我认为文献阅读应对大三同学作为选修，对大四同学作为必修。

首先，大三的专业课较多，与大量的文献阅读有所冲突。

其次，大三的同学可能对阅读的重要性和趣味还不甚了解，影响到了他们阅读的积极性。

再次，大三的同学专业知识不足，可能在阅读文献时遇到较多疑惑，产生畏难情绪。

总之，要使同学感受到阅读文献是一种享受才能使这门课达到最佳效果。

顺便提及一点对专业课的建议。

我认为分子生物学可以提到大一上学期进行，而细胞生物学或微生物可以提到大一下学期，而对大一的其他专业课进行适当的压缩。

我的建议是出于三点考虑。

一、分子、细胞知识与高中的生物学连接紧密，不需要高深的数理化知识，大一的同学可以接受。

二、与普通动植物生物学相比，这些课程更能代表当前的生物前沿。

许多生科的同学在大一就对生物失去了兴趣，很可能是因为大一的学习未能使他们看清生物研究的方向。

三、提早接触分子、细胞的知识，有利于大二大三时同学在实验室中的科研。

总而言之，这次实习加深了我对科研的了解和喜爱，增强了我对自己的信心，并将对我以后的研究学习产生极大的帮助。

我真心的喜欢和感谢这次活动。