Application of recombinant antibody Application of recombinant antibody library for screening specific library for screening specific

antigens in a bovine sperm cell antigens in a bovine sperm cell subpopulationsubpopulation

Zhou JingZhou Jing1070803710708037

1. Introduction1. Introduction1.1 Phage display techniques ( 噬菌体展示技术 ) 是一种基因表达产物和亲和选择相结合的技术 .

以改构的噬菌体为载体，把待选基因片段定向 插入噬菌体外壳蛋白质基因区，使外源多肽或 蛋白质表达并展示于噬菌体表面，进而通过亲 和富集法表达有特异肽或蛋白质的噬菌体 .

1985 年 Smith 首次证实丝状噬菌体 fd 基因组能通过基因工程的手段进行改造，将 EcoRⅠ 内切酶的部分基因片段 (171bp 和 132 bp与 pⅢ 基因融合，获得的重组噬菌体可在体外稳定增生，表达产物能被抗 EcoRⅠ 内切酶抗体所识别 .

George P. Smith

单链丝单链丝状噬菌状噬菌体展示体展示系统 系统

噬菌体展示系统分类

1.2 Introduction of this article Predetermining the sex of offspring is crucial in preventing X- linked disease transmission in humans and in increasing the rate of genetic progress in livestock. Separation of X- and Y-chromosome-bearing sperm for gender preselection is the obvious preference since sex determination takes place before fertilization.Many attempts to separate X and Y sperm have been described using physical parameters, such as swimming velocity, density, surface charge or sex-specific antigens.

Sorting by flow cytometry is accurate but inefficient ,as well as expensive. Antibody phage display is a new technology for the identification of novel target molecules. Antibody synthetic libraries are useful for the identification of self-antigens or phylogenetically conserved antigens in comparison to other methods based on immunization.

The present report describes efforts to identify plasma membrane proteins specific for eithermale or female cells on bovine sperm cells through an antibody phage display library.

2. Materials and methods2. Materials and methods2.1 Materials

Griffin.1 library——a human scFv phagemid library made from synthetic V-gene segments.

Antibodies——rabbit polyclonal anti-VCSM13 HRP conjugate antibodies;rabbit anti-scFv;Goat anti-rabbit IgG FITC

E.coli strains——E.coli TG1; E.coli HB2151

In this library, the VH and VL sequence connected with a spacer sequence was inserted into an NcoI-NotI restriction site and followed by the M13 gene III sequence in phagemid vector pHEN2.Recombinant scFv phages were rescued in Escherichia coli suppressor strain TG1 in the presence of helper phage VCSM13.

Dr. G. WinterCentre for Protein Engineering,

Medical Research Council, Cambridge, UK

James D. Marks et al., THE JOURNAL OF BIOLOGICAL CHEMISTRY,1992

By inserting an amberamber stop codonstop codon between the antibody gene and gene Ⅲ, when phage is grown in a supE suppressor strain of Escherichia coli, the amber codon is read as glutamine and the antibody fused to pIII is displayed on the surface of the phage. When the phage is grown in non-suppressor strains, the amber codon is read as a stop codon, and soluble proteinsoluble protein is secreted from the bacteria.

James D. Marks et al., THE JOURNAL OF BIOLOGICAL CHEMISTRY,1992

噬粒 (phagemid/phasmid)

由质粒载体和单链噬菌体载体构建成 既有质粒的复制起点，又有噬菌体的复制起点

以 M13 为例 : 在大肠杆菌细胞里，可以按正常的双链质粒分子进行复制，生成双链 DNA; 当宿主细胞受到辅助噬菌体感染后，在噬菌体基因Ⅱ蛋白质的作用下， 又可像单链噬菌体 M13 一样按滚环模型复制出单链 DNA ，然后由辅助噬菌体提供的外壳蛋白包装成噬菌体颗粒并在包装后释放出宿主细胞 .

许多噬菌粒利用 lac 启动子调控抗体 - PⅢ 融合产物的表达，而在抗体 -PⅢ 产物展示过程中，葡萄糖作为 lac 启动子的分解代谢抑制物，被转移或耗尽会影响产生单价噬菌体颗粒的融合产物的表达 .

辅助噬菌体 (helper phage)

一类自身 DNA 复制效率极低的突变型，但可为宿主细胞的质粒 DNA 提供的复制和包装所需的蛋白酶和外壳蛋白 .

2.2 Methods

1.Preparation of membrane

sperm proteins

2.Selection of phage antibodies on

membrane spermproteins

3.Production of soluble

monoclonal scFv

4.Screening of monoclonal scFv by flo

w cytometry

bovine sebovine semenmen

((Bos indicus, Nelore))

buffer Centrifugation

300*g,10minbuffer

Sonication

Centrifugation

Dialyzation

Lowry’s method(Protein concentrations)

1111

Immnunotubes

coatingIncubation

Washing

Elution

Neutralization

Helper phage VCSM13

Concentration

Precipitation

Griffitn.1 Griffitn.1 librarylibrary

2222

E.coli TG1

infe

ctioin

fectio

nn

ELISAELISA(rabbit polyclonal anti-VCSM13 HRP

conjugate)

3 rounds

E.coli HB2151 Agar (Amp,

G)

96-well plate 1

96-well plate 2

Adding Amp & IPTG when OD600~0.9

SDS-PAGE(scFv

concentration)

ELISAELISA(rabbit polyclonal anti-VCSM13 HRP

conjugate) Stocking (Glycerol ,-80℃)

Flow cytometryFlow cytometry Nelore (B. indicus) and Simental (Bos ta

urus) sperm cells, PBMCs of male and

female crossbred Zebu cattle

3333

4444

3. Results3. Results

Titration of eluted phage showed that there was significant enrichment in their number between the first and second rounds of panning, but not in the third round (Table 1). Eluted phages were used to infect E. coli TG1 and were rescued with VCSM13 helper phage.

3.1 Selection of phage antibodies anti-bovine cells

Screening of polyclonal phage antibodies showed that those from the second round of selection had the greatest positivity (Fig. 1).

On the other hand, there was small increase in the number of precipitated phages between the second and third rounds of panning (Table 1).

As a result, the phages produced in the second round were used to infect E. coli HB2151 and 89 clones of monoclonal soluble scFv were produced.

3.2 Screening of scFv anti-bovine sperm cells by flow cytometry

The supernatant of 89 clones containing soluble scFv was added. Previously, the average concentration of scFv in the culture supern

atant was estimated by SDS-PAGE as 11.7 μg/ml. The data obtained were organized into three groups according to n

umber of bound cells: 0–40, 40–60 and 60–100% (Table 2). Twenty-four clones bound to 40–60% of the sperm cells, while 58 cl

ones bound to <40% and seven clones bound to >60%. Subsequently, the 24 selected clones were analyzed for binding to

Nelore bovine sperm cells. Results were similar to the previous assay.

3.3 Screening of selected scFv clones binding to male and female leukocytes by flow cytometry

The scFv clones that bound to 40–60% of sperm cells were submitted to binding assay against male and female bovine leukocytes by flow cytometry. The same protocol for sperm cells was used for leukocytes.

Fig. 2 Screening of selected scFv clones binding to subpopulations of sperm cells and male and female leukocytes by flow cytometry. Sperm cells of Simental were treated with supernatant containing about 2.3 μg of scFv of clone H2 (B) or supernatant of HB2151 bacteria culture (A). PBMCs of male and female crossbred Zebu were also treated with supernatant of clone H2 (D and F, respectively) or HB2151 bacteria culture (C and E, respectively). Among 24 clones tested, only clone H2 bound to leukocytes.

Twenty-three clones did not bind to male and female leukocytes, but one clone bound to 22.9% of malemale cells (Fig. 2).

Male

Female

Sperm

4. Discussion4. Discussion

4.1 The results suggest that clone H2 recognizes one epi

tope on one subpopulation of leukocytes and sperm cells.

It can be hypothesized that our antibodies recognize a specific epitope present in subpopulations of leukocytes or cells in different stage of activation.

Thus Using an antibody phage display library, monoclonal antibodies against different sperm proteins were obtained in a less laborious and time-consuming manner.

4.2 The results also suggest the presence

of antigens characteristic of subpopulations of sperm cells and these antigens are apparently sex-specific.

4.3 It is speculated that differences in protein compo

sition between X- and Y-chromosome-bearing spermatozoa might occur for a minor component of themembranewith a low antigenicity and/or carbohydrate epitope.

Phage display provides an alternative technology for production of monoclonal antibodies against low antigenic molecules instesd of 2D electrophoresis or immunological techniques to identify proteins for sperm sexing.