Project

Abstract

Aflatoxin is a highly toxic substance which exists on grains. The most poisonous kind of Aflatoxin is Aflatoxin B1 (AFB1).

Since it is difficult to degrade the toxin, we decided to use ScFv (single chain variable region fragments), an antibody, to neutralize it. We use ScFv to neutralize AFB1 is because it had been used before and the molecular modification of it is easy.

We used ScFv, SH3 ligand and 6*his-tag to construct fusion protein so that it can combine with SH3 domain. In this way we can use cells to recover the ScFv that have been expressed out to reduce the antibody-antigen complex that exists on grains. We anchored SH3 domain on E.coli ’s cell membrane protein LGT to recover the ScFv.

Keywords: Aflatoxin B1 ScFv SH3 ligand SH3 domain LGT

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Background

Basic Theory of Aflatoxin B1

Aflatoxin is the assembly for a type of toxic secondary metabolities mainly produced by Aspergillus Flavus and Aspergillus parasiticus.¹ It widely exists in poultry feed, food and crops. Aflatoxin was designated as a Class 1 carcinogen by the World Health Organization (WHO) cancer research institutions --International Agency for Research on Cancer, IARC) . ²

Depending on the color shown under UV irradiation, Aflatoxin can be divided into G families (gives out green fluorescence, whose wavelength is of 450 micro-millimeters) and B families (gives out blue fluorescence, whose wavelength is of 425 micro-millimeters). The main body of Aflatoxin includes Aflatoxin B1, Aflatoxin M1, Aflatoxin G1 and Aflatoxin G2. Aflatoxin B1 is the most common one, and has the strongest biological reactivity. Its acute toxicity is ten times the potassium cyanide has, and 68 times the arsenic has.³

图片fig.1

fig.1：Structure formula of Aflatoxins

From the chemical structure point of view, Aflatoxins are very similar, ontaining C, H, O three elements. They are all the derivatives of dihydrofuran coumarin, contains one pair of the furan ring (bifuran), and an oxygen miscellaneous naphthalene O ketone (coumarin). The former is the basic structure of toxicity, the latter may be associated with carcinogenesis. The molecular weight of Aflatoxin is 312 ~ 346, mp 200 ~ 300 ℃.When melting, it wouid be decomposed. ⁴ Aflatoxin is insoluble in water, the maximum solubility in water is only 10ppm. ⁵

Aflatoxin is one of the most stable mycotoxins that have been found so far, crystalline Aflatoxin B1 is very stable, neither high-temperature (200 ℃) nor ultraviolet radiation can destroy it. It can decompose quickly in strong alkaline solution, but the reaction is reversible.⁶

Toxicity of Aflatoxin B1

The toxicity mechanism of Aflatoxin B1 will induce cancer and the toxicity of cells(Eaton and Gallaghen,1994). Briefly, when the Aflatoxins B1 enters the cells, it will become hydroxylated metabolin metabolized by monooxygenase in endoplasmic reticulum(ER), then they can be metabolized further into glucuronide and sulfate conjugates. It can also be oxidized into active epoxide. The epoxide can hydrolyze spontaneously into AFB1-8,9-dihydrodiol. They can also combine with protein which causes the toxicity of cells. This kind of epoxide can react with DNA or protein. Additionally, the epoxide can be detoxicated into Glutathione (GSH) – conjugate by inducible glutathione S-transferase.⁷

Researches on Toxicity to Tissue Culture Human Cells

1.According to Legator(etc.)’s report, 5ppm Aflatoxin can prevent human pneumonocyte from growing. 0.1ppm Aflatoxin can reduce 50％ of the mitosis of the pneumonocyte and make it turn into giant cell. It can also inhibit 45％ of the combination of DNA and 1.0ppm can inhibit 80％ of it.

2.Zuckerman discovered that tissue culture of stem cell heat with 10ppm Aflatoxin B1 will totally lose their RNA in cytoplasm. Chromatin in the nucleus will decrease obviously, too. Mix low concentration of Aflatoxin B1 with the cultivation of stem cells inhibits the combination of DNA and RNA

3.Related to Alessandro Weitz,etc.’s report, Aflatoxin B1 will damage the structure of the human lymphocytes which grows on phytohaemagglutinin(PHA). In the absence of PHA, Aflatoxin B1 will cause the dissolve of the human lymphocytes.⁸

According to the reports above, we can easily conclude that Aflatoxin B1is highly toxic and covers an extensive region. Thus, it is necessary to implement the treatment of Aflatoxin B1.

However，Aflatoxin B1 is stable in the condition of light, heat and acid. Only if it is heated to 280—300℃ that the Aflatoxin B1 will split. ⁹ Besides, both physical and chemical absorption will damage the nutritional value of food and feed. Therefore, we need to use biological method to cope with Aflatoxin B1. Due to the technical difficulty of biodegradation，we decided to utilize Scfv to reduce the toxicity of Aflatoxin B1.

The basic theory of ScFv(single chain variable fragment)

图片fig.2

Scfv is a new type of recombinant protein, fragment of antibody, which consists of the variable regions of heavy chain(VH) and light chain (VL) ,combining by a short linker peptide. It can spontaneously fold into native conformation, maintaining the specific and avidity of FV. It also have following advantages:

It has the function to neutralize toxin

It has small molecular weight and does not require glycosylation to form an useful antibody molecules so it is convenient to express in prokaryocyte.

It has strong penetration，thus it is easier to secrete

It doesn’t have the useless C-terminal of antibody which can not neutralize toxin

It is simple for molecular modification. ¹⁰ Molecular modification means making the expressed products easier to be detected and purified through adding his-tag label,etc at the C-terminal of ScFv, or constructing ScFv genes for fusion protein. The recombination protein neither affects ScFv combines with antigen, nor does harm to the biological activity of those protein which fused with it.¹¹

Previous iGEM Work Related to ScFV

Year&Teams	Project	ScFv target
2005 MIT2005 MIT	biosense	DNA
2008 Freiburg	Modular Synthetic	Receptor System Interfaced with Nano | Breadboard | TCV and Fragment
2009 Berkeley_Wetlab	Display-O-matic UC Benhclcy 2009	ESPA
2010 EPF_Lausanne	Asaia the pink force against Malaria	Pbs2l
2012 Potsdam_Bioware	Antibody Generation System	EGFR (epidermal growth factor receptor)
2012 Trieste	Jolly JoCare Cathelicidin	LL-37
2012 Tec-Monterrey Allergen	Apoptin&TRAIL	Content Cell | Content Cell
Content Cell	Content Cell	Content Cell

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Overview

clean, safe and efficient approach, we constructed our engineered bacteria to adhere to the Aflatoxin B1 in the environment, inspired by a traditional Chinese snack, Lyudagunr. When we segregate and collect the bacteria from the environment, the Aflatoxin B1 can be reduced.

Our engineered bacteria will achieve the following goals. First, the bacteria will express AFB1(Aflatoxin B1)’s ScFv. The ScFv will be excreted through the membrane of E. coli, to the liquid or semi-solid environment.

When existing AFB1 which is relevant to our AFB1-ScFv, the excreted AFB1-ScFv will be specifically bound with AFB1.

Meanwhile, the bacteria will express a type of engineered membrane protein, part of which exposed, and let it bind with AFB1-ScFv. Thus the bacteria are able to capture free AFB1-ScFv in the environment.

After a period of time, AFB1-ScFv can combine a massive amount of Aflatoxin B1 in the environment, which is immobilized, and adherent to the membrane of the engineered bacteria. Simultaneously will the toxicity of toxin be reduced.

图片fig.3

Because of the structural similarity, we named our engineered bacteria with a Chinese Snack, Lyudagunr.

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Design

In order to realize our initial proposition, we acquired the AFB1 -ScFv sequence information from NCBI(see table 1). （Shown in the figure 3(A)） According to relevant reference, PEI S, SUN D, GUO D succeeded in the expression and testing of anti-AFB1 ScFv in E. coli.¹² In our project, we modified and optimized the codon of ScFv. We fused the N-terminal of ScFv with a signal peptide (OmpA)¹³ and linked them with GLY-SER linker. The signal peptide can permeate through the membrane while target protein was translating, with the target protein downstream secreted through phospholipid bilayers. Also, the signal peptide was cut by enzymes in the cell and remained on the membrane, secreting target proteins.

Then, we fused the C-terminal of ScFv with 6＊his-tag. Proteins with such tag can be identified by specific antibodies and absorbed by Ni-NTA column for purification. Therefore, a 6＊his-tag can help us to detect protein expression.

Meanwhile we selected a pair of unitable protein perssad, fused on target ScFv and the membrane respectively, to collect ScFv altogether. Keasling proved that the eukaryotic ligand-domain system can be exerted well in microorganisms.¹⁴ So we chose one of the pairs mentioned by Keasling, SH3 ligand and SH3 domain for molecular construction. We fused SH3 ligand to the C-terminal of ScFv with GLY-SER linker, which can bind the target protein with any protein perssad which carries SH3 domain. Thus we finished the integral modification of ScFv. Last but not least, we utilized the LGT membrane protein constructed by 2012 SJTU-BioX-Shanghai[15]，whose N-terminal is outside the cell to construct a ScFv collector. We fused SH3 domain on the N-terminal of LGT so that it can bind with any biobricks with SH3 ligand outside the cell. Thus we finished the design of ScFv collector, whose structure is showed as fig.11.（Shown in the figure 3（B））

After the expression of the two artificial parts in E. coli, hopefully they can excrete ScFv, neutralize AFB1 and recycle AFB1 as planned. We will test them both in experimentation and mathematical modeling.

In conclusion， if we can finish all the design above，we can achieve the goal of the project.

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References

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