Method for synthesizing secreted human serum albumin employing bombyx mori posterior silk gland
A technology of human serum albumin and posterior silk gland, which is applied in the field of silkworm synthesis and secretion of exogenous proteins, can solve the problems of frequent allergic reactions and inability to purify products multiple times, so as to ensure biological safety, improve production efficiency and economical Benefits, the effect of reducing production costs
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Embodiment 1
[0026] A) The pBHSA plasmid construction method and steps are as follows:
[0027] According to the human serum albumin (Human serum albumin, HSA) gene sequence, design the upstream primer DDDDK-HAS-F that contains enterokinase restriction site to amplify human serum albumin, as shown in SEQ ID NO.17: 5'- gatgatgatgataaggatgctcacaagagtgaggt-3'; the downstream primer HAS-R is shown in SEQ ID NO.18: 5'-ttagagacctaaggcagcttgactt-3', the HSA gene is amplified by PCR technique. The HSA PCR reaction system is shown in Table 1, and the HSA PCR reaction conditions are shown in Table 2.
[0028] Table 1 HSA PCR reaction system
[0029] name
Element
PrimeSTAR HS Polymerase
0.25ul
5×PrimeSTAR Buffer
2.5ul
dNTP Mixture
2ul
Upstream primer DDDDK-HSA-F
1ul
downstream primer HSA-R
1ul
HSA plasmid
0.1ul
wxya 2 o
up to 25
[0030] Table 2 HSA PCR reaction conditions
[0031]
[0032]The PC...
Embodiment 2
[0045] A) The pBHSA plasmid construction method and steps are as in Example 1.
[0046] B) pBHSA plasmid ( figure 1 ) and a helper plasmid capable of providing piggyBac transposase ( figure 2 ) were mixed at a ratio of 1:1, the total concentration of the two plasmids was 0.5 μg / μl, the plasmids were dissolved in 0.5 mM phosphate buffer solution containing 5 mM sodium chloride at pH=7, and then introduced into silkworms to lay eggs by microinjection Into fertilized eggs within 5 hours afterward, the total volume of introduction is 5nl. The microinjected silkworm eggs were reared to adults at 25° C., 80% humidity, and 12 hours of light, and crossed with non-transgenic silkworms (G1 generation). During the turning green stage of the G1 generation eggs in the transgenic experiment, a transgenic silkworm moth expressing the DsRed marker gene was observed by a fluorescence microscope (Olympus, SZX12, Japan), containing 15 transgenic positive silkworms, and the silkworms were bred...
Embodiment 3
[0053] A) The pBHSA plasmid construction method and steps are as in Example 1.
[0054] B) pBHSA plasmid ( figure 1 ) and a helper plasmid capable of providing piggyBac transposase ( figure 2 ) were mixed at a ratio of 1:1, the total concentration of the two plasmids was 0.45 μg / μl, the plasmids were dissolved in 0.6 mM phosphate buffer solution containing 4 mM sodium chloride at pH=7, and then introduced into silkworms to lay eggs by microinjection Within 4 hours after fertilization, the total volume of introduction is 12nl. The microinjected silkworm eggs were reared to adults at 25° C., 85% humidity, and 12 hours of light, and mated with non-transgenic silkworms for subsequent generation (G1 generation). During the turning green stage of the G1 generation eggs in the transgenic experiment, one transgenic silkworm moth expressing the DsRed marker gene was observed by a fluorescence microscope (Olympus, SZX12, Japan), and two transgenic positive silkworms were obtained. T...
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