Application of recombinant Application of recombinant antibody library for antibody library for screening specific antigens screening specific antigens in a bovine sperm cell in a bovine sperm cell subpopulation subpopulation Zhou Jing Zhou Jing 10708037 10708037
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Application of recombinant antibody library for screening specific antigens in a bovine sperm cell subpopulation Zhou Jing 10708037.
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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
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.
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
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.