Expression Of Human Anti Hepatitis B Surface Antibody In Pichia Pastoris

today, a variety of molecular biology method has beenapplied gradually in the aspect of antibodies to transform, such asrecombinant DNA technology. Appeared to antibody fusion proteins, smallmolecules, antibody, humanized mouse represented by the sheetresistance and so on a series of genetic engineering antibody. This aseries of genetic engineering antibody, makes antibodies clinicalutility ratio increase quickly. However, humanized mouse singleantibodies different source resistance cannot be eliminated, and shorthalf-life in the body, many times using different levels of human antimouse antibody (HAMA); Antibodies and small molecule structure comparedto natural antibodies is incomplete, so it is difficult to show the FcPart mediated various biological effects. To solve these problems, thisarticle choose laboratory to have been built, based on the pichiaexpression vector pPICZa, for immune individuals (antibody of secondliver surface electropositive blood resistance) as the object, to builda complete human antibody heavy chain expression system (light chain)antibody expression library. In the antibody expression library,according to the Ag-Ab atopy, screening potential light chain and anti-HBs antibody heavy chain variable region sequence.The anti-HBs light chain and heavy chain antibodies in order to completeand summarized yeast, methanol induced after ELISA screening and western blot analysis, in contrast to anti-positive HBs immunoglobulinlibrary screening for light chain and heavy chain variable regionsequence, synchronous restructuring to complete antibody expressionvector pPICZ alpha IgG, transformation of building engineering bacteria,in order to analyze what kind of method for the restructuring of theanti-higher than HBs titer. The results prove that, in turn,restructuring method for anti-higher than HBs titer.The expression of human recombinant antibody of second liver surfaceresistance1. The two-step restructuringThe entire screening for hepatitis b surface antibody heavy chain intopichia cells after linearization, screening positive clones, and willmake characteristic for the cloning and then light chain to completethe hepatitis b surface antibody, build a complete antibody expressionsystem, methanol induction expression.2. One-step restructuringWill antibody screening for anti-HBs light chain variable region cDNAand heavy chain variable region cDNA and reorganization to the samepeople have built complete antibody expression vector pPICZ alpha IgG,transformed yeast cells after linearization, build a complete antibodyexpression system, methanol induction expression.A490, according to the results of both two step and step method,recombinant antibody in1,2days to express quantity is low, and0dayswithout Ming is different;3,4days express quantity increased; Peakday5,5-7antibody expression, no obvious changes into the plateau.The difference between the two is gained by the two-step recombinantantibody expression, to express the various periods were higher thanin synchronization method.The experimental results show that: the hepatitis b surface antibody full light chain and heavy chain in turn into pichia, gained by the amountof recombinant antibody expression than synchronous completerestructuring vitamin k, VH to the same human antibody expression vectorfor restructuring pPICZ alpha IgG antibody expression.Cloning and expression of anti-HBS antibody is anti-HBS antibodiesin the clinical application of laid the foundation, this article indetail further promoted the anti HBS antibodies to clinicalapplication.