Cdna Cloning Of Human Lysozyme In The Cho / Of Dhfr ~ - Cell Secretory Expression

This article mainly according to GenBank already the person bacteriolysin gene which announces, uses RT-PCR the method, succeeded from the person placenta organization expands adds and has cloned its DNA; Constructed the person bacteriolysin really nuclear secretion to express the carrier, dyed the Chinese hamster ovary cell using the fat altogether extension (CHO-/dhfr-), screening obtained expresses the person bacteriolysin the CHO resistance , to the time obtained the stable highly effective expression, produced the person bacteriolysin for the large-scale industrialization to lay the foundation.In tests one first acts according to in Genbank the person bacteriolysin gene mRNA sequ-ence, designs specificity using oligo6.0 software expands adds directs the thing; Separates from the fresh person placenta organization deputes total RNA, after two steps RT-PCR, first counter copies synthesizes first chain DNA, then uses the synthesis specially to direct the thing to carry on PCR to expand adds, the rubber recycling product links the enzyme with T4 to link it on the pMD18-T carrier, transforms into the DH5 alpha feeling condition cell, obtains the masculine clone. Further fragment cuts the link in pMD18-T on the carrier person bacteriolysin mature peptide DNA with restrictive enzyme EcoRV and the Xho1 double enzyme, again will recycle the target strip belt will link the really nuclear secretion to express on material particle pSec-Tag2/HygroB. Cut the appraisal after PCR and the double enzyme, the personbacterioly-sin gene success links expresses on the carrier. The sequence analysis result also showed that, exp-ands in the production increase to code the mature peptide 105th bit of alkali base to have the sudden change, in GenBank is A, this research expands adds is T, but because is located the th-ird bit of alkali base, GGU certainly does not affect the glycine (glysine) translation type. The-refore, after the translation obtained person bacteriolysin level of structures have not certainly changed.In the experimental second high school, the use fat technology, will contain the person bacteriolysin gene the pSecTag2/HygroB-hLYZ carrier and the pSV2/dhfr-2DNA material particle (3: 1) and fat according to 4: 6 proportions extensions dye in the dihydro folic acid return to original state enzyme flaw China hamster ovary cell CHO-k1/dhfr- cell. After adds the moist mildew element B antibiotic and the ammonia armor (MTX) and removes H, T (hypoxanthine, thymine deaeration nuclear glucoside) under double screening, has obtained 24 masculine cell clone body. RT-PCR confirms in the hLYZ gene already conformity like gene group, and has its mRNA copying. Stochastically chooses an expansion to raise and to use in exchange the non- seroculture under, pressurizes in MTX expands adds the DHFR gene; At the same time, the code person bacteriolysin gene sequence which dyes along with the DHFR altogether extension also obtains along with it expands adds, then enable the target protein expression quantity to obtain the enhancement, on the collection cell the clear nutrient fluid after the Ni+-Resin column purification. After the purification protein appraises its relative molecular mass and the theory value after SDS-PAGE and Western blot basically tallies. Explained the person bacteriolysin fusion protein expresses in the system in dihydro folic acid return to original state enzyme /CHO-k1 to achieve success the expression.